p27 and p21 in gene therapies

ABSTRACT

The expansion of a population of stem cells or progenitor cells, or precursors thereof, may be accomplished by disrupting or inhibiting p21 cip1/waf1  and/or p27, cyclin dependent kinase inhibitors. In the absence of p27 activity, progenitor cells move into the cell cycle and proliferate; whereas in the absence of p21 activity, stem cells move into the cell cycle and proliferate without losing their pluripotentiality (i.e., their ability to differentiate into the various cell lines found in the blood stream). Any type of stem cell or progenitor cell, or precursor thereof, including, but not limited to, hematopoietic, gastrointestinal, lung, neural, skin, muscle, cardiac muscle, renal, mesenchymal, embryonic, fetal, or liver cell may be used in accordance with the invention. The present invention provides a method of expanding a cell population, cells with decreased p27 and/or p21 activity, transgenic animals with a disrupted p27 and/or p21 gene, pharmaceutical compositions comprising the cells of the invention, and methods of using these cells in gene therapy (e.g., stem cell gene therapy) and bone marrow transplantation.

RELATIONED APPLICATIONS

[0001] The present application claims priority to co-pending provisionalapplications, U.S. Ser. No. 60/213,627, filed Jun. 23, 2000, and U.S.Ser. No. 60/188,120, filed Mar. 9, 2000, each of which is incorporatedherein by reference in its entirety.

GOVERNMENT SUPPORT

[0002] The work described herein was supported, in part, by grants fromthe National Institutes of Health (DK50234, HL44851, HL55718, DK02761,AI07387) and the U.S. Department of Defense. The United Statesgovernment may have certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Hematopoiesis is the process by which hematopoietic pluripotentstem cells mature into functional blood cells (i.e., red blood cells(erythrocytes), white blood cells (T-cells, B-cells, NK cells, dendriticcells, basophils, polymorphonucleated cells, macrophages, monocytes, andeosinophils), and platelets). In the current model of hematopoiesis, allblood cells begin as pluripotent stem cells. These pluripotent cells arepartitioned between resting and proliferating compartments, and duringhematopoiesis some of these cells are transformed to committedprogenitors of red blood cells, white blood cells, or platelets by theinfluence of multiple growth factors and cytokines. These committedprogenitor cells undergo further differentiation and commitmentinfluenced by growth factors and cytokines. The committed cells are alsopartitioned between resting and proliferating compartments; however,many more of these cells are proliferating. These committed progenitorcells give rise to morphologically identifiable immature precursor cells(i.e., blasts), which populate the marrow. These precursor cells maturefurther and eventually enter the blood where they are influenced furtherby growth factors and cytokines.

[0004] High levels of production of mature blood cells are needed toreplace their rapid turnover in the body (tens of billions of cells perday in the human with rapid increments during times of physiologicstress). Maintenance of blood cell production requires a highly cytokineresponsive progenitor cell pool with prodigious proliferative capacityand a smaller population of stem cells intermittently feeding daughtercells into the proliferative compartment. The proliferative activity ofthese very important hematopoietic stem cells has been hypothesized tobe highly restricted to prevent susceptibility to myelotoxic insult orconsumption of the regenerative cell pool (Mauch et al. Bone MarrowTransplant 4:601-607, 1989; Mauch et al. Int. J Radiat. Oncol. BiolPhys. 31:1319-39, 1995; Gardner et al. Exp. Hematol. 25:495-501, 1997;each of which is incorporated herein by reference). Once these stemcells embark on a path of high proliferation, they appear to surviveonly 1 to 3 months (Drize et al. Blood 88:2927-2938, 1996; incorporatedherein by reference). Hematopoietic tissue has therefore been thought tobe organized such that stem cells are relatively quiescent and cytokineresistant, but that their more differentiated offspring have extremelyrobust proliferative potential (Ogawa Blood 81:2844-53, 1993;incorporated herein by reference). The dichotomy of resistance toproliferative signals by stem cells and the brisk responsiveness byprogenitor cells is a central feature of hematopoiesis, and themolecular mechanisms governing it are not well understood.

[0005] Stem cells and progenitor cells are used in research, bone marrowtransplantation, and gene therapy; however, stem cell expansion withoutloss of multipotentiality is a problem. Current technology is based ondriving stem cells to proliferate with superphysiologic doses ofcytokines. These cytokines unfortunately have pleiotropic effects whichinclude differentiation of primitive cells. The result of thesetechniques is expanded cell numbers but a loss of multipotentiality. Dueto these problems in expanding stem cell populations, one third ofpatients are currently denied autologous bone marrow transplantationbecause of inadequate stem cell numbers. For example, cord blood stemcells are the best source of stem cells for minority groups, yet theyare inadequate in number to transplant adults.

[0006] Stem cell gene therapy has been a failure to date largely due tothe inability to achieve gene transfer in quiescent cells. Both bonemarrow transplantation and gene therapy would be revolutionized bysuccessful stem cell and progenitor cell expansion technology.

SUMMARY OF THE INVENTION

[0007] The present invention provides a method of expanding a populationof stem cells or progenitor cells by inhibiting p27 and/or p21 activityin the cells. The method comprises steps of providing at least one cell,in which the activity and/or amount of p27 and/or p21 within the cell isdecreased, and expanding the population of cells. Although cells of anyorigin or at any stage of differentiation may be used, hematopoieticstem or progenitor cells are preferred. Inhibition of p27 and/or p21 maybe accomplished by disruption of the gene encoding p27 and/or p21,inhibition of gene transcription, inhibition of translation of the p27and/or p21 mRNA, inhibition the kinase inhibiting activity of the p27and/or p21 protein, inhibition of the exogenous signals upregulating p27and/or p21, inhibition of signaling pathways controlling p27 and/or p21expression, etc. The present invention demonstrates that a decrease inp27 activity in the progenitor cells disrupts the dominantanti-proliferative tone that governs cell cycle entry by progenitorcells and induces cell proliferation. Similarly, the invention showsthat a decrease in p21 activity in the stem cells disrupts the dominantanti-proliferative tone that governs cell cycle entry by stem cells andpermits expansion of the stem cells without differentiation or withminimal differentiation or without loss of multipotentiality. Disruptingand/or inhibiting p27 and/or p21 allows for a highly specific and highlyfeasible strategy for permitting stem and/or progenitor cell expansionleading to the use of these cells in bone marrow transplants, genetherapy, or regeneration of other tissue types by pluripotent progenitorcells.

[0008] The present invention also provides a population of progenitorcells or stem cells containing a disrupted or inhibited p27 and/or p21.These cells may have decreased p27 and/or p21 activity when compared towild type cells, or the inhibition of p27 and/or p21 activity may haveonly been temporarily induced in order to expand the population and thecells may “re-gain” full p27 activity after the expansion. In certainembodiments, the p27 and/or p21 gene may be disrupted in thesecells—both copies or only one copy. The p27 and/or p21 gene may bedisrupted in the progenitor cell itself or in a precursor of theprogenitor cell (e.g., a stem cell). The present invention also providesa pharmaceutical composition comprising a therapeutically effectiveamount of p27-depleted progenitor cells or precursors thereof, and anoptional pharmaceutically acceptable excipient. In certain preferredembodiments, the inventive p27-depleted progenitor cells, or precursorsthereof, are also depleted for p21. The present invention also providesa pharmaceutical composition comprising a therapeutically effectiveamount of p21-depleted stem cells or progenitor cells, and an optionalpharmaceutically acceptable excipient. In certain preferred embodiments,the iventive p21 -depleted stem cells, or derivative thereof, are alsodepleted of p27.

[0009] The present invention also provides a transgenic animal with atleast one copy of the p21 and/or p27 genes altered. The genes may bealtered in all cells of the animal or in only a portion of the cells ofthe animal. In a preferred embodiment, the hematopoietic cells of theanimal have had at least one copy of the p21 and p27 genes altered.

[0010] In another aspect of the present invention, the cells of theinvention are used in bone marrow transplantation. In certain preferredembodiments, the genome of at least one cell has been altered by thehands of man, and these cells are optionally delivered to an animal orplant. In another preferred embodiment, cells in which the p21 activity,p27 activity, or both activities are decreased are administered to ananimal or plant.

[0011] In yet another aspect of the present invention, the cells of theinvention are used in gene therapy (e.g., stem cell gene therapy) ortissue regeneration. In a preferred embodiment, the p21 and/or p27 geneis altered in the administered cells. In another particularly preferredembodiment, the p21 and/or p27 activity is decreased compared to a wildtype cell. The genome of at least one cell may also be altered by thehands of man to correct a genetic defect, and these altered cells areoptionally delivered to an animal or plant. The genomic alterationwithin these cells may be accomplished before, after, or during theexpansion of the cell population. The genomic alteration may include,but is not limited to, insertion of an exogenous gene, mutation of anendogenous gene, repair of an endogenous gene, deletion of a gene, etc.

BRIEF DESCRIPTION OF THE DRAWING

[0012]FIG. 1. Distribution of G₀ vs. G₁ in the lineage negative bonemarrow mononuclear cell population defines an increased cycling fractionin p21 −/−mice. Mouse bone marrow cells were stained with lineageantibodies, pyronin Y (RNA dye), and Hoechst 33342 (DNA dye). Lineagenegative (Lin) cells were gated using a stringent parameter. Cellsresiding in G₀ appear at the bottom of the G₀/G₁ peak and G₁ cells arethe upper part as indicated (a). The average G₀% in lin-Hoechst^(low)cells from six experiments is shown in the graph (b). Data represent theMean+SE, n=6, p=0.005. 1-2 litter mates of each genotype were analyzedin each experiment.

[0013]FIG. 2. Response of p21 −/− mice to 5-FU treatment in vivodemonstrates a higher cycling status and increased sensitivity to toxicinjury. a. CAFC reduction after a 5-FU pulse. A single intravenousinjection of 5-FU at the dose of 200 mg/kg was performed, and cells forlong-term culture with limiting dilution were obtained one day after theinjection. CAFCs were counted at week 5. Y axis values=[(CAFCs fromun-treated mice−CAFCs from 5-FU treated mice)/CAFCs from un-treatedmice]×100%. Data represent the mean from three independent experiments.Three litter mates for each genotype were used in each experiment and3-5 limiting dilutions were applied for each sample. The student's testwas used to analyze the data (n=3, p=0.0019). b. Survival outcome aftersequential 5-FU treatment. 5-FU was administered i.p. weekly at dose of150 mg/kg, and the survival rates of the groups were defined. Resultswere analyzed using a log-rank nonparametric test and expressed asKaplan-Meier Survival curves (n=10, p=0.0054).

[0014]FIG. 3. Animal survival after serial bone marrow transplantation(BMT) demonstrates reduced self-renewal of hematopoietic potential. Malemice were used as marrow donors. Female recipient mice were lethallyirradiated with 10 Gy whole body irradiation (WBI) at 5.96 Gy/min. Twomillion nucleated cells were injected intravenously into the lateraltail veins of warmed recipients. Recipient mice were monitored daily forsurvival for more than one month. The mice were sacrificed after 2-4months, and bone marrow cells were prepared from those sacrificed andinjected into new female irradiated recipients. This process wasrepeated for an additional four times. a. Cumulative survival afterserial BMT. Each group included 10 mice initially. The donor marrow fromthe previous transplant was injected into a new recipient individuallyand therefore the actual recipient number was reduced during the serialtransplantation. The ratio between actual survival animal number at eachBMT and the total number at 1^(st)BMT is plotted as survival % (Y axisvalue). b. Radiation-protection of the marrow from the 4^(th)BMT. 5×10⁵cells from the 4^(th)BMT mice were transplanted into the lethallyirradiated recipients described as above and survival data were analyzedusing a log-rank nonparametric test and expressed as Kaplan-MeierSurvival curves (n=6/each group, p=0.002). Similar results were obtainedat lower doses (10⁵) of donor cells (n=10/each group, p=0.008, curve notshown). c. Donor contribution monitored by PCR. The contribution of theoriginal donor cells was monitored by a PCR-based semi-quantitativeanalysis for Y-chromosome specific sequence (Sry) using an aliquot ofmarrow sample from each transplant. DNA was prepared from donor cellscollected at the 4^(th) transplant and 200 mg was used for the PCRanalysis. 2% agarose gel was used to display the PCR products. Leftpanel in the left gel shows the positive controls which have been mixedwith male and female DNA at the ratios indicated. Complete contributionfrom donor cells was further confirmed by p21 genotyping PCR shown atthe right gel. Similar results were obtained from the 1^(st), 2^(nd),and 3^(rd) transplant (data not shown).

[0015]FIG. 4. p21−/− stem cell depletion is not due to altered bonemarrow homing. Donor bone marrow cells were stained with the cytoplasmicdye, CFSE, and intravenously injected into lethally irradiated mice.Bone marrow and spleen were harvested 9 hours after injection, andnucleated cells were stained with Sca-1 and lineage antibodies andanalyzed by flow cytometry. 2-3 litter mates of each genotype wereanalyzed in each experiment. Data shown is bone marrow cells from one oftwo experiments with similar results.

[0016]FIG. 5. CAFCs over the course of serial BMT confirm stem cellexhaustion. Long-term culture with limiting dilution was performed onthe donor cells of each transplant to quantify the frequencies ofhematopoietic progenitors and stem cells. Normal, not-transplantedmarrow was used as a control to assure the quality of the stroma and thecomparability of the experiments at different times. Data arerepresented as the Mean±S.D. and graphed as log scales in Y axis. All pvalues are less than 0.05 (−/−vs. +/+). a. CAFCs at week 5 from the1^(st) and the 3^(rd) transplant. b. CAFCs at the indicated weeks fromthe 4^(th) transplant.

[0017]FIG. 6. The long-term culture and colony forming assaysdemonstrate an unchanged stem cell pool size and an enlarged progenitorpool in the p27 −/−mice. (a) comparison of CAFCs scored at week 5between p27 +/+ and −/−mice (per harvest of two femurs). Each data pointwas generated from three to five limiting dilutions. Each pair waspooled from two to three −/− or +/+ littermate mice in each experiment.Data were analyzed using the paired t-test (P=0.3861, n=7). (b)Comparison of CFCs between p27 +/+ and −/− mice (per harvest of twofemurs). Data represent colony-forming ability at day 10. Each pair waspooled from two to three −/− or +/+ littermate mice in each experiment.Each data point was generated from four replicates, and data wereanalyzed using the paired t-test (P−0.0006, n−5). Each line shows onedata pair from the same experiment, and the bold thicker line shows theaverage value from all the independent experiments.

[0018]FIG. 7. Altered cell cycle profile of progenitor cells, but notstem cells, in the p27 −/− marrow. Mouse bone marrow cells were stainedwith lineage antibodies and stem cell marker (Sca-1) to separate theenriched stem (Sca-1⁺Lin⁺) and progenitor (Sca-1⁺Lin⁺) pools (a, upperpanel). Simultaneous staining with the DNA dye, To-pro-3, was used todetermine the percentage of S+G2/M cells in each population (a, middleand lower panels). Data from multiple experiments are summarized in (b)(P=0.0215, n=7 in Sca-1⁺Lin⁺ cells; P=0.3591, n=9 in Sca-1⁺Lin cells).To determine the ratio of G0 to G1 in stem cell population, the RNA dye(pyronin Y, PY) and DNA dye (Hoechst 33342) were used instead ofTo-pro-3, and the percentage of G0 (py^(low)) was obtained in the G0/G1fraction (Hoechst^(low)Lin) 4,28 shown in (c) (P=0.1591, n=7). Each datapoint represents the mean from one to three −/−or +/+ littermate mice ineach experiment. Data were analyzed using the paired t-test.

[0019]FIG. 8. Treatment with 5-FU in vivo demonstrates more active cellcycling in the progenitor pool, but not in the stem cell pool of the p27−/−mice. One day after a single intravenous injection of 5-FU at thedose of 200 mg/kg was given, cells for long-term culture with limitingdilution and colony forming ability were obtained. CAFCs were counted atweek 5, and CFCs were counted at day 10. y-axis values=[(CAFCs or CFCsfrom untreated mice− CAFCs or CFCs from 5-FU treated mice)/CAFCs or CFCsfrom un-treated mice]×100%. Data represent the mean from multipleindependent experiments. Three littermates for each genotype were usedin each experiment, and three to five limiting dilutions were used foreach sample in the long-term culture. The Student's t-test was used forcomparative analysis: ^(*)P=0.0044, n=5 for CFC and P=0.2852, n=6 forCAFC, comparing p27−/− (black bars) with p27 +/+ (white bars) cells.

[0020]FIG. 9. Serial bone marrow transplantation (BMT) demonstrates anunaltered self-renewal of hematopoietic stem cells and an enhancedactivity of progenitor cells in the p27−/− transplanted mice. Male micewere used as marrow donors. Female recipient mice were lethallyirradiated with 10 Gy whole-body irradiation at 5.96 Gy/min. Two millionnucleated cells were injected intravenously into the lateral tail veinsof warmed recipients. Recipient mice were monitored daily for survivalfor more than one month. The mice were euthanized after one to fourmonths, and bone marrow cells were prepared from those euthanized andinjected into new female irradiated recipients. This process wasrepeated four more times. a. CAFC decline (relative to pre-BMT sample)over the course of serial BMT. The donor cells of each transplant weresubjected to long-term culture with limiting dilution to quantify thefrequencies of stem cells. Normal, not-transplanted marrow was used as acontrol to assure the quality of the stroma and the comparability of theexperiments at different times. There was no significant differencebetween the p27−/− (solid line) and p27+/+ (dotted line) groups. b. CFCactivities during serial BMT indicate expansion of progenitor pools inthe p27−/− transplanted mice. The Student's t-test was used foranalysis: *P=0.001 in the third BMT and P>0.05 in other BMTs betweenp27−/− (black bars) and p27+/+ (white bars) cells. c. Short-termradiation protection of the marrow from the 4^(th) transplant. 10⁵ cellsfrom the fourth BMT mice were transplanted into the lethally irradiatedrecipients described as above, and survival data were analyzed using alog-rank nonparametric test (P=0.036, n=10 in the p27−/− group (solidline) or n=9 in the p27+/+ group (dotted line)) and expressed asKaplan-Meier survival curves.

[0021]FIG. 10. Competitive repopulation assay demonstrates preferentialoutgrowth of p27−/− progenitor and mature cells following long-termengraftment. Equal numbers of bone marrow nucleated cells from p27+/+and p27−/− mice (five mice for each genotype) were mixed andtransplanted into five lethally irradiated recipients. Blood wascollected at 6, 9, and 11 months for semiquantitative p27 genotyping PCRanalysis. (a). At 11-12 months, mice were sacrificed and bone marrownucleated cells were prepared for PCR analysis (b) and hematopoieticcell culture. Individual colonies from CFC culture or individual CAFCand LTC-ICs from different wells were harvested and analyzed by PCR forp27 to determine the distribution of p27−/− (black bars) or p27+/+(white bars) in the indicated compartment (c). (d) Overall observedresults from this study compared with the conventionally expectedresult: p27−/− (black bars); p27+/+ (white bars).

[0022]FIG. 11. p21-antisense reduces the G₀ fraction of transduced CD34+cord blood cells. In six independent experiments, transduction ofp21-antisense decreased the proportion of cells in G₀ in the CD34⁺38⁻subpopulation of transduced CD34⁺ cordblood cells (7.3% p21-AS-V vs.16.4% GFP-V; p=0.007.

[0023]FIG. 12. Antisense-p21 increases primitive CFU-mix withoutaltering total CFC in transduced CD34⁺ and CD34⁺38⁻ cord blood cells.(a) The colonies generated by cells expressing p21 -antisense showed ahigher proportion of colonies with myloid and erythroid cells (CFU-mix)representing more primitive hematopoietic cells than colonies of thecontrol vector transduced cells (CD34⁺: 9.3 vs. 2.8 colonies/600 cells,p=0.02; CD34⁺38⁻: 19.2 vs. 7.1 colonies/600 cells, p-0.002). (b)Transduced cells were plated four days after the beginning oftransduction in semisolid CFC-medium. Neither CD34⁺(n=4) nor CD34⁺38⁻cells transduced with p21 -antisense showed an altered total colonynumber compared to cells transduced with the control vector.

[0024]FIG. 13. Antisense-p21 expands LTC-IC as assessed by limitingdilution analysis. CD34⁺ and CD34⁺ 38 ⁻ cells transduced withp21-antisense gave rise to a significantly higher number of long-termculture initiating cells (LTC-ICs) compared with cells transduced withthe control vector, indicating a higher proportion of stem cells in thep21 -antisense transduced cell population (CD34⁺: 33.5 vs. 19.3LTC-ICs/100000 cells (p=0.04); CD34⁺38⁻: 416 vs. 228 LTC-ICs/100000 cell(p=0.03))

[0025]FIG. 14. p21^(cip1) anti-sense enhances human CD34⁺ cellengraftment of NOD/SCID mice. FIG. 14 demonstrates the percent of humancells detectable in the blood of animals transplanted with cells exposedto either control (GFP-V) or p21-anti-sense encoding (p21-AS-V) vector.

Definitions

[0026] “Animal”: The term animal, as used herein, refers to human aswell as non-human animals, including, for example, mammals, birds,reptiles, amphibians, and fish. Preferably, the animal is a mammal(e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, or apig), most preferably a human. An animal may be a transgenic animal.

[0027] “Decreased p21 activity”: The phrase “decreased p21 activity”refers to a decreased amount of p21 protein or mRNA transcript in thecell and/or a decreased level of p21 activity. The decreased p21activity may be accomplished by any method known in the art includingsmall molecule inhibitors of p21, antisense agents, aptamers, geneknockout, antibodies, overabundance of the countervailing cyclin D/CDK4complex to overwhelm p21 inhibition, etc. Preferably, the reduction inactivity is at least 50% when compared to wild type cells. Morepreferably, the reduction is at least 75% or 90%, and most preferably,decreased p21 activity refers to an undetectable level of p21 activity.

[0028] “Decreased p27 activity”: The phrase “decreased p27 activity”refers to a decreased amount of p27 protein or MRNA transcript in thecell and/or a decreased level of p27 activity. The decreased p27activity may be accomplished by any method known in the art includingsmall molecule inhibitors of p27, antisense agents, aptamers, geneknockout, antibodies, overabundance of the countervailing cyclin E/CDK2and/or cyclin A/CDK2 complexes to overwhelm p27 inhibition, etc.Preferably, the reduction in activity is at least 50% when compared towild type cells. More preferably, the reduction is at least 75% or 90%,and most preferably, decreased p27 activity refers to an undetectablelevel of p27 activity (e.g., by Northern analysis, Western analysis,enzymatic assay, etc.).

[0029] “Homologous” or “homologue”: The term “homologous”, as usedherein is an art-understood term that refers to nucleic acids orpolypeptides that are highly related at the level of nucleotide or aminoacid sequence. Nucleic acids or polypeptides that are homologous to eachother are termed “homologues.”

[0030] The term “homologous” necessarily refers to a comparison betweentwo sequences. In accordance with the invention, two nucleotidesequences are considered to be homologous if the polypeptides theyencode are at least about 50-60% identical, preferably about 70%identical, for at least one stretch of at least 20 amino acids.Preferably, homologous nucleotide sequences are also characterized bythe ability to encode a stretch of at least 4-5 uniquely specified aminoacids. Both the identity and the approximate spacing of these aminoacids relative to one another must be considered for nucleotidesequences to be considered homologous. For nucleotide sequences lessthan 60 nucleotides in length, homology is determined by the ability toencode a stretch of at least 4-5 uniquely specified amino acids.

[0031] “Peptide” or “Protein”: According to the present invention, a“peptide” or “protein” comprises a string of at least three amino acidslinked together by peptide bonds. Inventive peptides preferably containonly natural amino acids, although non-natural amino acids (i.e.,compounds that do not occur in nature but that can be incorporated intoa polypeptide chain; see, for example,http://www.cco.caltech.edu/˜dadgrp/Unnatstruct.gif, which displaysstructures of non-natural amino acids that have been successfullyincorporated into functional ion channels) and/or amino acid analogs asare known in the art may alternatively be employed. Also, one or more ofthe amino acids in an inventive peptide may be modified, for example, bythe addition of a chemical entity such as a carbohydrate group, aphosphate group, a farnesyl group, an isofarnesyl group, a fatty acidgroup, a linker for conjugation, functionalization, or othermodification, etc.

[0032] “Polynucleotide” or “oligonucleotide”: Polynucleotide oroligonucleotide refers to a polymer of nucleotides. The polymer mayinclude natural nucleosides (i.e., adenosine, thymidine, guanosine,cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, anddeoxycytidine), nucleoside analogs (e.g., 2-amninoadenosine,2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine,5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine,2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine,C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine,2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine), chemicallymodified bases, biologically modified bases (e.g., methylated bases),intercalated bases, modified sugars (e.g., 2′-hydroxylribose,2′-fluororibose, ribose, 2′-deoxyribose, and hexose), or modifiedphosphate groups (e.g., phosphorothioates and 5′-N-phosphoramiditelinkages).

[0033] “Progenitor cell”: “Progenitor cell” refers to unipotent ormultipotent, committed or determined cells derived from stem cells.Progenitor cells undergo further differentiation and commitment to giverise to morphologically identifiable immature cells. Progenitor cellswhich may be used in accordance with the present invention includehematopoietic, neural, mesenchymal, gastrointestinal, muscle, cardiacmuscle, kidney, skin, lung, and embryonic progenitor cells. In certainpreferred embodiments, hematopoietic progenitor cells are positive forthe CD34 marker. In another preferred embodiment, hematopoieticprogenitor cells are positive for Sca-1 and lineage markers (Spangrudeet al. “Purification and characterization of mouse hematopoietic stemscells [published erratum appears in Science 1989 June 2;244 (4908):1030]” Science 241:58-62, 1988; incorporated herein by reference).Progenitor cells may be identified morphologically, kinetically, oroperationally as further described below in the definition of a stemcell.

[0034] “Stem cell”: “Stem cell” refers to any pluripotent cell thatunder the proper conditions will give rise to a more differentiatedcell. Stem cells which may be used in accordance with the presentinvention include hematopoietic, neural, mesenchymal, gastrointestinal,muscle, cardiac muscle, kidney, skin, lung, and embryonic stem cells. Togive but one example, a hematopoietic stem cell can give rise todifferentiated blood cells (i.e., red blood cell (erythrocyte), whiteblood cell (T-cell, B-cell, neutrophil, basophil, eosinophil, monocyte,macrophage), or platelet) or neural or muscle cells. In terms ofmorphology, hematopoietic stem cells are small mononuclear cellsnormally found in the bone marrow of adults. These cells can be mobileand can also be found in the blood at a concentration of 1-5 per 10⁵nucleated cells. During development, hematopoietic stem cells may befound in various locations in the body including the liver, spleen,thymus, lymph nodes, yolk sac, blood islands, and bone marrow.

[0035] Stem cells can also be characterized by their ability (1) to beself-renewing and (2) to give rise to further differentiated cells. Thishas been referred to as the kinetic definition.

[0036] Also, an operational definition of stem cell regards the stemcell as a colony-forming unit in various laboratory systems. Forexample, when suspensions of bone marrow (i.e., hematopoietic) cells areinjected intravenously into heavily irradiated mice in which the spleenand marrow are reduced to stroma and are hematologically empty, discretemacroscopic colonies of cells are observed in the animal's spleen after8-10 days.

[0037] A cell that meets any one of these three definitions of stem cellis considered to be a stem cell according to the present invention.

[0038] “Therapeutically effective amount”: The term “therapeuticallyeffective amount” refers to the amount of an agent needed to elicit thedesired biological response. In the present invention, the agent iscells (e.g., stem cells, progenitor cells, etc.). The therapeuticallyeffective amount of stem cells in a bone marrow transplant, for example,is enough cells to repopulate the bone marrow space and rescue thepatient from aplastic anemia. In the case of gene therapy, thetherapeutically effective amount of cells is the amount necessary tocorrect the recipient's underlying genetic defect. In the case of tissuedamage or degeneration, the therapeutically effective amount of cells isthe amount necessary to improve the function or structure of theabnormal tissue.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

[0039] The present invention provides a system for expanding apopulation of progenitor or stem cells under the condition of decreasedp21 and/or p27 activity. These cells may then be used in pharmaceuticalcompositions in tissue transplants, bone marrow transplants, and/or genetherapy (e.g., stem cell gene therapy).

[0040] p21^(cip1/wafl)

[0041] p21^(cip1/waf1) is a cyclin-dependent-kinase inhibitor (CKI).CKIs participate in the sequential activation and inactivation ofcyclin-dependent kinases central to progression through 25 the cellcycle (Sherr Cell 79:551-555, 1994; Sherr et al Genes Dev 9:1149-63,1995; each of which is incorporated herein by reference). Specifically,the p21 gene has been found to play a key role in regulating themovement of cells from the G₀ to the G₁ stage of the cell cycle andtherefore regulates the entry of stem cells into the cell cycle. Byperturbing these regulatory circuits in stem cells the proliferation ofstem cells is changed.

[0042] Targeted disruption of the p21 gene in mice resulted in cellswhich are impaired in their ability to achieve cell cycle arrestfollowing irradiation (Brugarolas et al. Nature 377:552-7, 1995; Deng etal Cell 82:675-84, 1995; each of which is incorporated herein byreference), and antisense p21 has been shown to release humanmesenchymal cells from G₀ (Nakanishi et al. Proc. Natl. Acad. Sci. USA92:4352-6, 1995; incorporated herein by reference). Therefore, p21 playsa role in at least some cell types in the transition out of the cellcycle and maintenance in G₀. However, in hematopoiesis, levels of p21have not been shown to be increased in CD34⁺ cells (Taniguchi et al.Blood 93:4167-4178, 1999; Yaroslavskiy et al Blood 93:2907-2917; 1999;each of which is incorporated herein by reference), and p21−/− mice havenot been noted to have an altered hematologic profile (Brugarolas et al.Nature 377:552-557, 1995; Deng et al. Cell 82:675-84, 1995; each ofwhich is incorporated herein by reference). Further, bone marrowprogenitor cells from p21−/− mice paradoxically have decreasedproliferative ratios in response to cytokines (Mantel et al. Blood88:3710-9b, 1996; Braun et al. Blood Cells Mol. Dis. 24:138-148, 1998;each of which is incorporated herein by reference). However, we foundhigh levels of p21 mRNA when we assessed the quiescent stem cell-likefractions of bone marrow mononuclear cells. We therefore propose,although we do not wish to be bound by any particular theory, that p21plays distinct roles in the subcompartments of the hematopoieticcascade-p21 seems to augment progenitor cell proliferation and toinhibit stem cell proliferation. By inhibiting the activity of p21,populations of hematopoietic stem cells are able to grow withoutinhibition (Cheng et al. “Hematopoietic Stem Cell Quiescence Maintainedby p21^(cip1/waf1) ” Science 287:1804-1808, Mar. 10, 2000; incorporatedherein by reference).

[0043] p21 as used in the present invention can refer to either the geneand/or protein form of p21 or any homolog of p21, as will be clear fromcontext. The homolog should be at least 50% homologous to the mouse p21DNA or protein sequence, preferably 60% homologous, and more preferably70% homologous. A homolog of p21 may also be identified by its activitysuch as being a cyclin dependent kinase inhibitor. In another preferredembodiment, the homolog of p21 is identified by its location in thegenome (e.g., location on the chromosome).

[0044] The protein p27 is a member of the cyclin dependent kinaseinhibitor family, which includes p21, p27, and p57. p27 is molecularlydistinct from p21 in its carboxy terminus, interacts with similar butnot identical cyclin dependent kinases, and lacks p53 regulatedexpression (Polyak et al. “p27kip1, a cyclin-Cdk inhibitor, linkstransforming growth factor-beta and contact inhibition to cell cyclearrest” Genes Dev 8:9-22, 1994; Sherr et al. “Inhibitors of mammalian G1cyclin-dependent kinases” Genes Dev 9:1149-63, 1995; Sherr “Cancer cellcycles” Science 274:1672-7, 1996; each of which is incorporated hereinby reference). p27 acts by binding to and inhibiting the activation ofcyclin E-Cdk2 and cyclin A-Cdk2 complexes. Characterization of the p27protein and cloning and sequencing of the gene encoding the p27 proteinare described in detail in PCT application (WO PCT/US95/07361),incorporated herein by reference.

[0045] Disruption of the p27 gene in mice led to a phenotype markedlydifferent than the one of p21−/− mice in that its body habitus is largerwith hyperplasia of most organs (including hematopoietic organs), itspontaneously generates benign pituitary tumors, and it is infertile(Kiyokawa et al. “Enhanced growth of mice lacking the cyclin-dependentkinase inhibitor function of p27(Kip1)” Cell 85:721-732, 1996; Fero etal “A syndrome of multiorgan hyperplasia with features of gigantism,tumorigenesis, and female sterility in p27 (Kip 1)-deficient mice” Cell85:733-744, 1996; Nakayama et al. “Mice lacking p27(Kip1) displayincreased body size, multiple organ hyperplasia, retinal dysplasia, andpituitary tumors” Cell 85:707-720, 1996; each of which is incorporatedherein by reference). Like p21, however, p27 is associated withpost-mitotic differentiation in some cell types (Asiedu et al. “Complexregulation of CDK2 during phorbol ester-induced hematopoieticdifferentiation” Blood 90:3430-3437, 1997; Liu et al. “Transcriptionalactivation of the human p21 (WAF1/CIP1) gene by retinoic acid receptor.Correlation with retinoid induction of U937 cell differentiation” J.Biol Chem. 271:31723-31728, 1996; Kranenburg et al. “Inhibition ocyclin-dependent kinase activity triggers neuronal differentiation ofmouse neuroblastoma cells” J Cell Biol. 131:227-234, 1995; each of whichis incorporated herein by reference) and antisense p27 can suppress cellcycle arrest in mesenchymal cells (Coats et al. “Requirement of p27Kip1for restriction point control of the fibroblast cell cycle” Science272:877-880, 1996; Rivard et al. “Abrogation of p27Kip1 by cDNAantisense suppresses quiescence (GO state) in fibroblasts” J Biol. Chem.271:18337-18341, 1996; each of which is incorporated herein byreference).

[0046] Unlike p21, p27 is controlled by both translational andposttranslational mechanisms (Hengst et al. “Translation control ofp27Kip1 accumulation during the cell cycle” Science 271:1861-1864, 1996;Pagano et al. “Role of the ubiquitin-proteasome pathway in regulatingabundance of the cyclin-dependent kinase inhibitor p27 [see comments]”Science 269:682-685, 1995; each of which is incorporated herein byreference). A role for p27 in hematopoiesis is supported by direct flowcytometric evidence for expression in primitive cells (Tong et al.“TGF-βsuppresses cell division of Go CD34+cells while maintainingprimitive hematopoietic potential” Exp. Hematol. 26:684, 1998;incorporated herein by reference), expression in more mature progenitors(Taniguchi et al. “Expression of p21 (Cip1/Waf1/Sdi1) and p27(Kip1)cyclin-dependent kinase inhibitors during human hematopoiesis” Blood93:4167-4178, 1999; Yaroslavskiy et al. “Subcellular and cell-cycleexpression profiles of CDK-inhibitors in normal differentiating myeloidcells” Blood 93:2907-2917, 1999; each of which is incorporated herein byreference) and indirectly by improved retroviral transduction in thecontext of anti-sense p27 (Dao et al. “Reduction in levels of thecyclin-dependent kinase inhibitor p27(kip 1) coupled with transforminggrowth factor beta neutralization induces cell-cycle entry and increasesretroviral transduction of primitive human hematopoietic cells [InProcess Citation]” Proc. Natl. Acad. Sci. USA 95:13006-13011, 1998;incorporated herein by reference). As shown in the Examples below,disruption of the p27 gene allows for expansion of a population ofprogenitor cells in vivo as well as ex vivo. In terms of gene therapy, aminority population of stem cells with less than wild type p27 activitytends to predominate the progenitor and mature blood cell compartmentswithout leading to leukemia and polycythemia.

[0047] p27 as used in the present invention can refer to either the geneor protein form of p27 or any homolog of p27, as will be clear fromcontext. The homolog should be at least 50% homologous to the mouse orhuman p27 DNA or protein sequence. A homolog of p27 may also beidentified by its activity such as being a cyclin dependent kinaseinhibitor. In another preferred embodiment, the homolog of p27 isidentified by its location in the genome (e.g., location on thechromosome).

[0048] Stem Cells/Progenitor Cells

[0049] The stem cells or progenitor cells used in the present inventionmay be obtained from any tissue of an animal or plant at any stage ofdevelopment. The cells may be derived from lung, gastrointestinal tract,liver, kidney, neural, skin, muscle, cardiac muscle, bone, mesenchymal,or hematopoietic tissue. In certain embodiments of the presentinvention, the cells are obtained from embryonic or fetal tissues. Thecells may not be committed to a single tissue or may be able to producedifferentiated cells in a variety of tissues such as lung andgastrointestinal tract, or skin and neural tissue. Cells obtained fromembryonic or fetal tissues would be expected to have a greater potentialthan most cells found in adult animals.

[0050] The cells may be obtained from any animal or plant. Preferably,the animal is a mammal, and humans are particularly preferred. When thecells are to be eventually used in treating a patient, the stem cellsare preferably obtained from the same species as the animal to betreated. More preferably, the cells are obtained from the patient or aclose relative. In the most preferred embodiment, the cells areautologous. In another preferred embodiment, the stem cells are obtainedfrom a cell culture or tissue culture. These cells may be from a donoror from established cell lines. The stem cells utilized in the presentinvention can be purified using any available method such as, forexample, fluorescence-activated cell sorter (FACS) analysis,immunomagnetic bead purification (or other immunoprecipitation method),or functional selection using anti-metabolites, dye exclusion,resistance to cytokines, or attachment to lectins or other bindingmolecules.

[0051] In a particularly preferred embodiment of the present invention,the cells are hematopoietic cells. The hematopoietic cells may beobtained from any source. Preferably the cells are obtained from thehematopoietic tissue of an animal. Preferably the animal is a mammal.More preferably the mammal is a rat, mouse, rabbit, guinea pig, ferret,dog, cat, etc. In other preferred embodiments, the mammal is a human.The bone marrow is typically the site where hematopoietic stem cells orprogenitor cells are found; however, the spleen, liver, thymus, lymphnodes, umbilical cord blood, and blood may also be used to obtain thecells. The cells utilized in the present invention can be purified usingany available method such as, for example, fluorescence-activated cellsorter (FACS) analysis, immunomagnetic bead purification (or otherimmunoprecipitation method), or functional selection usinganti-metabolites.

[0052] When the stem cells are to be used in a bone marrow transplant orin stem cell gene therapy, the stem cells are preferably obtained fromthe same species as the animal to be treated. More preferably, the cellsmay be obtained by a biopsy such as a bone marrow biopsy from thepatient or a close relative. In the most preferred embodiment, the cellsare autologous. In another embodiment, the stem cells may be obtainedfrom a cell culture or tissue culture. These cells may be from a donoror from established cell lines.

[0053] The cells used in the present invention may be pluripotenthematopoietic stem cells or may be cells committed to one particularlineage. For example, the cell may be committed to erythropoiesis,granulopoiesis, or thrombopoiesis. A pluripotent cell may give rise toall types of blood cells including erythrocytes, platelets(thromobocytes), polymorphonucleated neutrophils, monocytes,macrophages, eosinophils, basophils, mast cells, B-cells, plasma cells,and T-cells. A myeloid stem or progenitor cell is more committed thanthe pluripotent cells and is only able to give rise to erythrocytes,platelets (thrombocytes), polymorphonucleated neutrophils, monocytes,macrophages, eosinophils, basophils, and mast cells. A lymphoid stem orprogenitor cell only gives rise to T-cells, B-cells, and plasma cells.

[0054] Inhibition of p21 and/or p27

[0055] Any method may be used in the current invention to decrease theactivity or amount of p21 and/or p27. These methods include, but are notlimited to, altering the transcription, translation, and/or enzymaticactivity of p21 or p27. Examples of altering the transcription includedisrupting the gene, altering the regulatory sequence of the p21 or p27gene (e.g., promoter, enhancer), adding antisense p21 or p27 agents tothe cells, etc. The splicing of the primary transcript may also bechanged by altering the splicing signals of the transcript. Thetranslation may be affected by altering the Shine-Delgarno sequence ofthe transcript, altering the codon usage in the mRNA transcript, addingantisense p21 or p27 agents to the cells, etc. The enzymatic activity ofthe p21 or p27 protein may be altered, for example, by contacting theprotein with a known inhibitor, contacting the protein with an antibodyknown to inhibit p21 or p27 activity, adding an allosteric effector,inhibiting post translational modifications of the protein, etc. Also,the complexes to which p21 or p27 binds or the kinase that it inhibitsand that can overwhelm its inhibitory function can be altered to createthe same net effect as is seen with decreased p21 or p27 activity.

[0056] When p21 or p27 activity is inhibited in the cell, the stem orprogenitor cell can enter the cell cycle and proliferate expanding thepopulation of cells. Preferably, the inhibition may be stopped ordecreased after the population of cells has been expanded to the desirednumber.

[0057] In a preferred embodiment, the p21 or p27 activity of the cell isinhibited by an agent known to inhibit the kinase inhibiting activity ofp21 or p27. These agents may be peptides, aptamers, proteins,non-peptide chemical compounds, polynucleotides, carbohydrates, etc.These inhibitors of p21 or p27 activity are preferably specific for p21or p27 and do not interact with and/or inhibit other proteins (e.g.,cyclin-dependent-kinase inhibitors) in the cells. In a particularlypreferred embodiment, these agents are hydrophobic enough to diffusethrough the plasma membrane of the cells or are taken up by the cells.Alternatively, the agents may be delivered into the cell viamicroinjection or lipofection.

[0058] In another preferred embodiment, the inhibitory agent is anantibody, or fragment thereof. Preferably, the antibody is specific forp21 or p27. The antibody's may bind to any location on p21 or p27 aslong as the binding of the antibody results in a decrease in p21 or p27activity. A preferred binding site on p21 or p27 includes the activesite (i.e., where the p21 or p27 protein binds to the kinase). Theantibody may be a whole antibody, a fragment, an antibody conjugated toanother agent, etc. Preferably, the antibody is monoclonal. In anotherpreferred embodiment, the antibody has been engineered to get into thestem cell or progenitor cell.

[0059] Antisense therapy is meant to include, e.g., administration or insitu provision of single-or double-stranded oligonucleotides or theirderivatives which specifically hybridize, e.g., bind, under cellularconditions, with cellular mRNA and/or genomic DNA encoding p21 or p27,or mutants thereof, so as to inhibit expression of the encoded protein,e.g., by inhibiting transcription and/or translation (Crooke “Molecularmechanisms of action of antisense drugs” Biochim. Biophys. Acta1489(1):31-44, 1999; Crooke “Evaluating the mechanism of action ofantiproliferative antisense drugs” Antisense Nucleic Acid Drug Dev.10(2):123-126, discussion 127, 2000; Methods in Enzymology volumes313-314, 1999; each of which is incorporated herein by reference). Thebinding may be by conventional base pair complementarity, or, forexample, in the case of binding to DNA duplexes, through specificinteractions in the major groove of the double helix (i.e., triple helixformation) (Chan et al. J Mol. Med. 75(4):267-282, 1997; incorporatedherein by reference).

[0060] In certain preferred embodiments, the antisense construct bindsto a naturally-occurring sequence of a p21 or p27 gene which, e.g., isinvolved in expression of the gene. These sequences include, e.g., startcodons, stop codons, and RNA primer binding sites.

[0061] An antisense construct of the present invention can be delivered,e.g., as an expression plasmid or viral vector which, when transcribedin the cell, produces RNA which is complementary to at least a uniqueportion of the cellular mRNA which encodes a p21 or p27 protein. Analternative is that the antisense construct is an oligonucleotide probewhich is generated ex vivo and which, when introduced into the cellcauses inhibition of expression by hybridizing with mRNA and/or genomicsequences of a p21 or p27 gene. The antisense construct may beintroduced into the cell via microinjection, lipofection, passivediffusion, or facilitated transport by chaperone molecules or moleculessuch as HIV tat, or peptides thereof (Schwarze et al. Science285:1569-1572, 1999; incorporated herein by reference). Sucholigonucleotide probes are preferably modified oligonucleotides whichare resistant to endogenous nucleases, e.g., exonucleases and/orendonucleases, and are therefore stable in vivo. Exemplary nucleic acidmolecules for use as antisense oligonucleotides are phosphoramidate,phosphothioate, o-methylated, and methylphosphonate analogs of DNA (U.S.Pat. Nos.5,176,996; 5,264,564; and 5,256,775; each of which isincorporated herein by reference). Additionally, general approaches toconstructing oligomers useful in antisense therapy have been reviewed(van der Krol et al. Biotechniques 6:958-976, 1998; Stein et al. CancerRes. 48:2659-2668, 1988; each of which is incorporated herein byreference).

[0062] Pharmaceutical Composition

[0063] The cells of the present invention may be used in apharmaceutical composition. The cells may have a normal to decreased p21and/or p27 activity. The cells may be used in immunotherapy, bone marrowtransplants, tissue transplants, or gene therapy (e.g., stem cell genetherapy). An exemplary pharmaceutical composition comprises atherapeutically effective amount of cells, optionally combined with apharmaceutically-acceptable excipient. The stem cells, progenitor cells,or other cells may or may not have been transfected, transformed, orinfected. In certain preferred embodiments, the cells have altered bythe hands of man.

[0064] The pharmaceutical compositions of the present invention may beadministered by any known method including, for example, intravenous,intramuscular, subcutaneous, intrasternal, intraosseous, and parenteraladministration. A more preferable mode of administration is parenteraladministration, and the most preferred method of parenteraladministration is intravenous administration. Injectable preparations,for example, sterile injectable aqueous or oleaginous suspensions may beformulated according to the known art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution, suspension or emulsion in anontoxic parenterally acceptable diluent or solvent, for example, as asolution in 1,3-butanediol. Among the acceptable vehicles and solventsthat may be employed are water, Ringer's solution, U.S.P. and isotonicsodium chloride solution. In addition, sterile, fixed oils areconventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil can be employed including synthetic mono ordiglycerides. In addition, fatty acids such as oleic acid are commonlyused in the preparation of injectables.

[0065] Transgenic Animal or Plant

[0066] The present invention provides for transgenic animals and plantswith alterations of the p21 and/or p27 gene(s). In a particularlypreferred embodiment of the present invention, the transgenic animal orplant has at least one copy of the p21 gene altered in at least one ofthe cells of the organism. In other embodiments, both copies of the p21gene have been altered. The present invention provides for a transgenicanimal or plant with at least one copy of the p21 gene and the p27 genealtered (i.e., mutated, changed, or disrupted) in at least one of thecells of the organism. In other embodiments both copies of the p21 geneand/or the p27 gene have been altered. The transgenic organism of thepresent invention may also have other genomic alterations in addition tothe p21 and/or p27 mutations. The term transgenic animal or plant ismeant to include an organism that has gained new genetic material fromthe introduction of foreign DNA, i.e., partly or entirely heterologousDNA, into the DNA of its cells; or introduction of a lesion, e.g., an invitro induced mutation, e.g., a deletion or other chromosomalrearrangement into the DNA of its cells; or introduction of homologousDNA into the DNA of its cells in such a way as to alter the genome ofthe cell into which the DNA is inserted, e.g., it is inserted at alocation which differs from that of the natural gene or its insertionresults in a knockout. The organism may include a transgene in all ofits cells including germ line cells, or in only one or some of itscells.

[0067] In certain embodiments, the transgenic animal or plant has a p21and/or p27 transgene, or fragment or analog thereof. In certain otherembodiments, the transgenic animal has a knockout for the p21 and/or p27gene. In a preferred embodiment of the present invention, at least oneof the hematopoietic stem or progenitor cells of the transgenic animalhas at least one copy of the p21 and/or p27 gene that has been changed,mutated, or disrupted.

[0068] Gene Therapy

[0069] The stem cells, progenitor cells, or other cells of the presentinvention with less than wild type p27 activity and, optionally lessthan wild type p21 activity, may be particularly useful in gene therapy,bone marrow transplants, and tissue repair/regeneration. In certainpreferred embodiments, the cells to be administered in a pharmaceuticalcompositions suitable for gene therapy have had their genomes altered bythe hands of man in order to correct a genetic defect or to insert atransgene. Given the ability of a minority population of p27−/− stemcells to predominate in the progenitor and mature blood cellscompartments as described below in the Example 2, the number of cellsneeded to improve the signs and symptoms of the genetic defect ispreferably far less than is normally required in stem cell gene therapy.This ability of p27−/− cells to expand in vivo and compete out wild typecells or cells without the desired alteration allows one to treatdiseases that require a high efficiency of gene transfer and/or geneeffect (e.g., sickle cell anemia). In a particularly preferredembodiment, the number of cells with the corrected genetic defect orwith the transgene inserted is less than 50% of the total number ofcells transplanted. More preferably, the number of cells is less than75% of the total number of transplanted cells, and most preferably, thenumber of cells is less than 90% of the total number of cellstransplanted.

[0070] In another particularly preferred embodiment, the number oftransplanted cells with altered genomes is sufficient to allow the cellpopulation to expand in vivo and compete out other wild type cells. Thisallows one to transplant a small number of cells initially, but have thecells derived from the transplanted cells dominate the cell populationin the transplanted individual after a set amount of time (e.g., weeks,months, years depending on the cell type being transplanted). In certainpreferred embodiments, the percentage of cells derived from thetransplanted cells and/or having the desired alteration in their genomemay reach at least 50%, 75%, 80%, 90%, 95%, or 99% of that particularcell population in the transplanted individual.

[0071] In a particularly preferred embodiment, both the activities ofp27 and p21 are less than the activities found in wild type cells. Thedecreased p21 activity allows for proliferation in the stem cellcompartment while the decreased p27 activity allows for proliferation inthe progenitor cell compartment. In another preferred embodiment, theinhibition of the activity for one or both of the cyclin-dependentkinase inhibitors is temporary.

[0072] In another particularly preferred embodiment, cells from tissuesother than hematopoietic tissue may be used in the regeneration of otherorgans or tissues. For example, neural cells may be used to regenerateneural tissues, or pluripotent stem cells may be used to regenerate anumber of different tissues.

[0073] These and other aspects of the present invention will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the inventionbut are not intended to limit its scope, as defined by the claims.

EXAMPLES Example 1 Hematopoietic stem cell quiescence in vivo ismaintained by p21^(cip1/waf1) and is critical for preventing prematureexhaustion of hematopoiesis

[0074] Methods

[0075] Generation of homozygous mice. Heterozygote 129/SV p21 +/− mice(Brugarolas et al. Nature 377:552-7, 1995; incorporated herein byreference) were obtained from the laboratory of Tyler Jacks (MIT,Boston) under the permission of the Subcommittee on Research Animal Careof the Massachusetts General Hospital (MGH). The mice were housed insterilized microisolator cages and received autoclaved food and drinkingwater at the MGH animal care facility. The inbred 129/SV heterozygotes(+/−) were bred to yield homozygous and wild-type offspring. The littermates from the same +/− parents were used in each experiment.

[0076] Mouse genotyping. Genotyping was achieved by DNA PCR. Briefly,genomic DNA was isolated from tail biopsy and analyzed by amplificationusing three primers: p21+116F (AAG CCT TGA TTC TGA TGT GGG C), p21 -135(TGA CGA AGT CAA AGT TCC ACC G), and Neo19+(GCT ATC AGG ACA TAG CGT TGGC). p21+116F was involved in the amplification of both mutant andwild-type alleles. The conditions for thermocycling were as follows:

[0077] Step 1: 94° C. for 4 min.

[0078] Step 2: 94° C. for 1 min.

[0079] Step 3: 64° C. for 1 min.

[0080] Step 4: 72° C. for 2 min.

[0081] Step 5: Repeat step 2-4 forty times

[0082] Step 6: 72° C. for 2 min.

[0083] Diagnostic mutant and wild-type band were 750 bp and 900 bp,respectively, by 2.0% agarose gel electrophoresis.

[0084] Bone marrow sampling. Mouse bone marrow was obtained from8-12-week-old animals from each group (−/−, +/+) sacrificed with CO₂.The marrow cell suspensions were flushed from femurs and tibias,filtered with 100-mesh nylon cloth (Sefar America, Inc., Kansas City,Mo.), and stored on ice until use.

[0085] Flow cytometric analysis. Flow cytometry was used to quantify thecell cycle status in the stem cell compartment. Bone marrow nucleatedcells were labeled with biotinylated anti-lineage antibodies (CD3, CD4,CD8, B220, Gr-1, Mac-1 (Caltac), TER-119 (Pharmingen)) andstreptavidin-FITC, then incubated with 1.67 ,μmol/L DNA dye, Hoechst33342 (Hst), and 1 μg/ml RNA dye, Pyronin Y (PY), at 37° C. for 45minutes, respectively. Flow cytometry was performed on FACSVantageInstruments (Becton Dickinson).

[0086] Colony forming assay. Bone marrow nuclear cells were cultured in0.8% methylcellulose, 30% fetal bovine serum, 1% bovine serum albumin0.1 mM 2-mercaptoethanol, 2.0 mM L-glutamine of α-MEM semi-solid matrixculture medium (StemCell Technologies, Inc.) with cytokine combinationsof 50 ng/ml hu-SCF (R & D System Inc.), 20 ng/ml mu-IL-3 (Genzyme), 20ng/ml hu-IL-6 (Genzyme), 50 ng/ml hu-G-CSF (R & D System Inc.), 20 ng/mlhu-GM-CSF (R & D System Inc.), and 2 U/ml hu-Epo (Amgen). Cells wereplaced at 20,000/200 μl/well into 48-well plates and placed at 37° C.,5% CO₂. At day 10, myeloid and erythroid colonies were scored, totaled,and reported as CFCs.

[0087] Long-term culture with limiting dilution. To quantify the stemcells, we adapted the cobblestone area forming cell (CAFC) assay(Ploemacher et al. Blood 74:2755-2763, 1989; incorporated herein byreference) with minor modifications as follows. To prepared stromallayers, murine bone marrow nucleated cells were cultured at 33° C. inlong term culture (LTC) medium (α-MEM with 12.5% mouse serum, 12.5%fetal bovine serum, 0.2 mM 1-inositol, 20 mM folic acid, 10⁻⁴ M2-mercaptoethanol, 2 mM L-glutamine, and 10⁻⁶ M hydrocortisone). After 2weeks, confluent stromal layers were trypsinized, irradiated (15 Gy),and subcultured in 96-well flat-bottomed plates at a density of2.5×10⁴/well. Cultures were then seeded with serially dilutedsingle-cell suspensions of femoral marrow in the same medium. Marrowpooled from two to ten animals of each type was seeded at 2-folddilutions (10⁵ - 1562 cells/well) for nucleated bone marrow cells.Cultures were very gently re-fed with 50 μl medium after semidepletionweekly and the CAFCs and/or blast colonies (Ploemacher et al. Blood74:2755-2763, 1989; Muller-Sieburg et al. J Exp. Med. 183:1141-50, 1996;incorporated herein by reference) were scored until the 6th week.

[0088] 5-FU exposure in vivo. The anti-metabolite, 5-FU, was used toselectively deplete cycling cells in vivo. 5-FU was administered i.p.weekly at a dose of 150 mg/kg and the survival rates of the groupsdefined. To more specifically test the effect of p21 on hematopoieticcells, we transplanted bone marrow cells from p21−/− or p21+/+ animalsinto lethally irradiated mice of the same genetic background (p21+/+,129/SV, 8 weeks old, Jackson), allowing the hematopoietic and immunesystem to repopulate for one month, and then observed the 5-FU effectson those animals. To test the sensitivities of primitive hematopoieticcells, a single injection of 5-FU i.v. at a dose of 200 mg/kg wasadministered and cells for long-term culture with limiting dilution wereobtained one day after the injection.

[0089] Serial bone marrow transplantation. Serial bone marrowtransplantation (Harrison et al. J Exp. Med. 147:1526-1531, 1978;Harrison Blood 55:77-81, 1980; Harrison et al. J Exp. Med.156:1767-1779, 1982; Harrison et al. J Exp. Med. 172:431-437, 1990; eachof which is incorporated herein by reference) was used to evaluate theability of stem cells to self-renew. Male mice (8-12 weeks old) wereused as marrow donors and the marrow cells prepared as above. Femalerecipient mice (8-10 weeks old, 129/SV, Jackson laboratory) werelethally irradiated using a Mark 1-Model 25¹³⁷ Cesium Irradiator (JLShepherd and Associates, San Fernando, Calif.) with 10 Gy whole bodyirradiation (WBI) at 5.96 Gy/min. One to two million nucleated cells in1 ml 199 Medium were injected intravenously through 27 gauge needlesinto the lateral tail veins of warmed recipients. Recipient mice weremonitored daily for survival for more than 30 days in which stem cellshave been noted by others to account for hematopoietic recoveryfollowing lethal irradiation (33). The mice were sacrificed at 2-4months and bone marrow cells were prepared from those sacrificed miceand injected into new female recipients. This process was repeated for 4sequential transplants with survival frequency plotted for each group.Long-term culture with limiting dilution described above was performedon the donor cells of each transplant to quantify the frequencies ofhematopoietic progenitors and stem cells.

[0090] Semi-quantitative DNA PCR for Y-chromosome. The contribution ofthe original donor cells was monitored by a PCR-based semi-quantitativeanalysis for Y-chromosome specific sequence (Sry) (Muller et al.Development 118:1343-51, 1993; incorporated herein by reference) usingan aliquot of each marrow sample described below. Briefly, DNA from bonemarrow cells was isolated using a Puregene kit (Gentra System, Inc.,Research Triangle Park, N.C.) according to the manufacturer'sinstruction. 200 ng of DNA was applied to the PCR reaction. Thesequences for the PCR primers are as follows (5′-3′): Sry primers: TCATGA GAC TGC CAA CCA CAG and CAT GAC CAC CAC CAC CAC CAC CAA; myogeninprimers: TTA CGT CCA TCG TGG ACA GC and TGG GCT GGG TGT TAG TCT TA. ThePCR cycles were: 10 min 94° C., 35 cycles of 94° C. for 10 sec, 65° C.for 30 sec followed by 5 min 72° C. A linear relationship between theratios of male genomic DNA to the total amount of DNA and the signalintensities of the PCR product was plotted simultaneously in order toquantify the contribution of donor cells.

[0091] Homing assay. Donor marrow nucleated cells were stained with thecytoplasmic dye, CFDA SE (CFSE) according to the manufacturer'sinstruction (Molecular Probes, Eugene, Oregon) and 2×10⁷ CFSE stainedcells were injected into lethally irradiated recipient mice. Spleen andbone marrow were harvested 9 hours after injection and cells stainedwith lineage markers and anti-Sca-1 antibody prior to flow cytometricanalysis.

[0092] Statistical analysis. Results from survival experiments wereanalyzed using a log-rank nonparametric test and expressed asKaplan-Meier Survival curves (Grzegorzewski et al. J Exp Med180:1047-57, 1994). The significance of the difference between groups inthe in vitro culture were evaluated by analysis of variance followed bya two-tailed Student's test.

[0093] Results

[0094] The direct impact of p21 on the stem cell compartment wasassessed using mice engineered to be deficient in p21. The cell cyclingstatus of stem cells was determined using the RNA dye, pyronin Y (PY),as a measure of quiescence among the lineage negative (lin-) (Spangrudeet al. Science 241:58-62, 1988; incorporated herein by reference) andHoescht 33342 (Ho)^(low) staining bone marrow cells (Leemhuis et al.Exp. Hematol. 24:1215-1224, 1996; incorporated herein by reference).Cells from p21−/− animals consistently demonstrated a smaller fractionin the PY low portion of the continuum (FIG. 1) (p=0.005, n=6)suggesting that p21 does function to impede the entry of stem cells intoactive cell cycle. Independently, rhodamine (Rho), a mitochondrial dye,and Ho were used to define the population of cells with low levels ofmetabolic activity and exclusion of Ho corresponding to a quiescent stemcell pool (Wolf et al. Exp. Hematol. 21:614-22, 1993; incorporatedherein by reference). The Rho^(low)/Ho^(low) population of lin ⁻Sca-1⁺cells was also smaller in the p21−/− animals (p=0.07, n=3) confirmingthis observation.

[0095] To further define this issue, we pulsed −/− or +/+ mice with 200mg/kg of the anti-metabolite, 5-fluorouracil (5-FU), to selectively killcycling cells (Berardi et al. Science 267:104-108, 1995; Lemer et al.Exp. Hematol. 18:114-118, 1990; each of which is incorporated herein byreference). Marrow was harvested one day after 5-FU injection and longterm co-culture or cobblestone area-forming cell (CAFC) assaysperformed. These assays linearly correlate with in vivo repopulatingpotential (Ploemacher et al. Blood 78:2527-2533, 1991; Ploemacher et al.Blood 74:2755-2763, 1989; each of which is incorporated herein byreference) and were used here as a stem cell assay instead ofcompetitive repopulation assays given the lack of a congenic mousestrain with 129/SV background. A significant reduction of CAFCs wasnoted after a pulse of 5-FU in the −/− group compared with the +/+ groupcontrols (60.5% vs. 10.8%, p=0.0019) (FIG. 2a).

[0096] When the animals were given 5-FU weekly as a challenge to assessthe relative restriction on cell cycle entry of primitive cells, thesurvival percentage in the −/− group was much lower than in litter mate+/+ controls (10% vs 70% in one month, p=0.0054) (FIG. 2b). To excludethe possible influence of toxicity to other tissues, we repopulated thehematopoietic system of lethally irradiated +/+ hosts with either +/+ or−/− bone marrow cells. One month after transplantation, we challengedthe reconstituted animals with an identical protocol of sequential 5-FUtreatment. A similar relative survival pattern was observed with micecarrying the −/− hematopoietic system demonstrating markedly increasedmortality compared with those with a +/+ hematopoietic system(p=0.0089). Therefore, death was due to hematopoietic and not othertissue sensitivity to antimetabolite treatment. Thus p21 restricts theentry of stem cells into cycle and protects hematopoietic cells fromdestruction by cell cycle dependent myelotoxic agents.

[0097] We next sought to determine whether the lack of p21 resulted inan increase in stem cell number in the basal state or a decline due tomore rapid depletion. The relative number of stem cells present in wildtype versus p21−/− mice was directly measured by limit dilution CAFCassays. A significant increase in primitive cells in the p21−/− animals(Table 1, p=0.0393, n=7) was noted. Thus, p21 provides a dominantnegative effect which is sufficient to inhibit stem cell cycling. In theabsence of p21 , the inhibition is alleviated, leading to an expansionof the primitive cell pool under resting conditions. In contrast, nosignificant differences in colony forming cells, bone marrow cellularityand white blood cells were noted (Table 2, 3, and 4) implying that p21has a differentiation stage specific function in hematopoietic stemcells. The paradoxical pro-proliferative effect of p21 in more matureprogenitors observed by others, may balance the inhibitory influence p21enforces on stem cells (Mantel et al. Blood 88:3710-9b, 1996; Braun etal. Blood Cells Mol Dis. 24:138-148, 1998; each of which is incorporatedherein by reference). This apparent dichotomy may reflect the complexbiochemical role p21 has been noted to play as either requisiteparticipant in cyclin-CDK complex formation necessary for movement ofthe cell through late G₁ into S, or as CKI inhibiting entry into S phase(LaBaer et al. Genes Dev. 11:847-862, 1997; incorporated herein byreference). We speculate that p21 plays a central role in determiningthe known differential sensitivity of stem cells versus progenitor cellsto proliferative stimuli and that the p21 effect represents a uniquelybimodal functional distinction between these two broad classes of cells.TABLE 1 Comparison of CAFCs scored at week 5 between p21 +/+ and −/−mice (per 10⁵) demonstrates increased stem cell numbers. Each pair waspooled from 2-3 −/− or +/+ litter mate mice in each experiment. Eachdata point was generated from 3-5 limiting dilutions and data wasanalyzed using the paired t-test. P21 +/+ p21 −/− Experiment 1 2.88 4.98Experiment 2 2.41 5.29 Experiment 3 0.83 1.06 Experiment 4 0.58 0.92Experiment 5 0.18 0.49 Experiment 6 0.87 1.33 Experiment 7 0.32 1.25Mean 1.15 2.19 p value, paired t-test 0.0393

[0098] TABLE 2 Comparison of CFCs between p21 +/+ and −/− mice (per 10⁴)indicates no difference in progenitors. Data represents colony formingability at day 10. Each pair was pooled from 2-3 −/− or +/+ litter matemice in each experiment. Each data point was generated from at least 4replicates and data were analyzed using the paired t-test. P21 +/+ p21−/− Experiment 1 66.65 50.00 Experiment 2 76.25 35.00 Experiment 3 60.0045.00 Experiment 4 55.00 80.00 Experiment 5 51.84 56.50 Experiment 642.33 35.67 Experiment 7 32.67 35.00 Mean 53.38 46.61 p value, pairedt-test 0.1755

[0099] TABLE 3 Comparison of total cell number per bone marrow harvestbetween p21 +/+ and −/− mice indicates no difference in cellularity.Each data point represents the mean from 1-3 −/− or +/+ litter mate micein each experiment. total cell number (× 10⁷/femur pair) was countedfrom each harvest and data were analyzed using the paired t-test. P21+/+ p21 −/− Experiment 1 1.4  1.3  Experiment 2 1.58 1.76 Experiment 32.5  2.08 Experiment 4 1.5  1.5  Experiment 5 1.63 1.27 Experiment 61.29 2.14 Mean 1.63 1.68 p value, paired -t-test 0.8284

[0100] TABLE 4 Comparison of blood cell counts between p21 +/+ and −/−mice (n = 10, Mean ± SD) indicates no significant difference in maturecell popula- tions. Blood was collected by tail bleeding. All the bloodcounts were per- formed and analyzed using the t-test for two sampleswith the same variance. WBC (× 10³/ul) RBC (× 10^(6/)ul) PLT (× 10³/ul)p21 +/+ 5.38 ± 2.95 9.08 ± 0.86 444.60 ±  55.99 p21 −/− 7.48 ± 1.83 9.38± 0.96 510.10 ± 141.65 p value 0.0670 0.2290 0.0950

[0101] The expansion of stem cells under normal homeostatic conditionsmay or may not reflect a capacity to self renew under conditions ofstress. The cytokine milieu dramatically changes during stress,including the elaboration of cytokine such as GM-CSF, G-CSF, orinterleukin-3 with strong pro-differentiative properties. Wehypothesized that the outcome of enhances proliferation of the stem cellcompartment under such conditions will markedly differ from normalhomeostasis and directly assesses stem cell self-renewal capacity usinga serial transplantation approach (Harrison et al J. Exp. Med.147:1526-1531, 1978; Harrison Blood 55:77-81, 1980; Harrison et al. J.Exp. Med. 156:1767-1779, 1982; Harrison et al. J. Exp. Med. 172:431-437,1990; each of which is incorporated herein by reference). Bone marrowfrom ten male animals in each genotype was individually transplantedinto lethally irradiated female mice. Two to four months afterengraftment, 1-2×10⁶ bone marrow cells from the transplanted recipientswere used as donor cells for a lethally irradiated host and the sameprocedure was repeated sequentially. Recipient animals began to dieafter the 3_(rd) serial transplant and marked differential survival inthe group was noted (FIG. 3a). No −/− transplanted animals survivedafter the 5^(th) transplant whereas the +/+ transplanted animals has a50% survival one month after the transplant. To confirm the paucity ofstem cells in −/− transplanted mice, we used two different doses ofcells from the 4^(th) transplant to rescue lethally irradiated hosts.The two irradiation protection experiments at different doses confirmedthe significantly poorer ability to rescue irradiated mice using cellsfrom the −/− group (FIG. 3b). We observed approximately 100%contribution from the original donor p21−/− or +/+ cells in hostsexamined after each transplant by semi-quantitative Y-chromosomespecific (Sry) PCR and p21 genotyping PCR (FIG. 3c).

[0102] To evaluate whether the transplantation data could be affected byaltered homing of stem cells in the absence of p21, we directly measuredthe localization of ex vivo fluorescently labeled p21−/− and +/+ bonemarrow cells with carboxy fluorescein diacetate succinimidyl diester(CFSE) (Holyoake et al. Blood 94:2056-2064, 1999; incorporated herein byreference) following transplantation. The fraction of mononuclear cellsor lin, Sca-1⁺ cells homing to either bone marrow or spleen was the samefor the −/− and +/+ mice (FIG. 4).

[0103] These functional in vivo parameters of stem cell function werecorroborated with quantitative in vitro measures of function of theprimitive cell compartment. CAFCs at week 5 from −/− mice werecompletely exhausted after the 3_(rd) transplant while detectable CAFCswere still noted in the +/+ group (FIG. 5a). Although an absence ofCAFCs after 4 weeks in both of +/+ and −/− groups from the 4 ^(th)transplant, early cobblestones (week 2 and 3) reflecting short-termrepopulating cells (Ploemacher et al. Blood 78:2527-2533, 1991;Ploemacher et al. Blood 74:2755-2763, 1989; each of which isincorporated herein by reference), demonstrated a significant difference(FIG. 5b). The CAFCs in −/− transplant recipients dampened to zero aftertwo weeks while the CAFCs in +/+ transplant recipient remaineddetectable at 3 weeks. The levels of CAFCs in −/− mice were alsosignificantly lower than in the +/+ group (all p values <0.05, FIG. 5b).

Example 2 Molecular boundary between stem and progenitor cells is markedby distinct CDKI dominance

[0104] Methods

[0105] Generation of homozygous mice. Heterozygote 129/B6 p27+/− micewere obtained from the laboratory of Dr. Andrew Koff (Sloan KetteringCancer Center, New York) under the permission of the Subcommittee onResearch Animal Care of the Massachusetts General Hospital (MGH). Micewere housed in sterilized microisolator cages and received autoclavedfood and drinking water at the Massachusetts General Hospital animalcore facility. The heterozygotes (+/−) were crossbacked into 129/svbackground and bred to yield homozygous and wild-type offspring. Thelittle mates from the same +/− parents were used in each experiment.

[0106] Mouse genotyping. Genotyping was achieved by DNA PCR. Briefly,genomic DNA was isolated from tail biopsy and analyzed by amplificationusing three primers (the sequences were provided by Dr. Andrew Koff):SW40 (5′-TCA AAC GTG AGA GTG TCT AAC GG3′), SW41 (5′ ACG GGC TTA TGA TTCTGA AAG TCG-3′) and SW39 (5′-ATA TTG CTG AAG AGC TTG GCG G-3′). SW40 isa forward primer that binds the region nt4-nt26 of pLambda-KIP-34-1.Used in conjunction with SW41, a reverse primer binding to nt209-nt186of pLambda-KIP-34-1, this will produce a PCR product of 206 bps from thewild-type locus. SW40 used in conjunction with SW39, a forward primerbinding to nt 1420-nt1441 of PMC1POLA, will produce a PCR product of 298bps from the mutant locus. All three primers were used together in thesame reaction to detect wild type and mutant loci. The conditions forthermocycling were as follows: Step 1: 94 ° C., 4 min; step 2: 90° C.,30 seconds, 55° C., 30 seconds, 72° C., 1 min, for 35 cycles; step 3:72° C., 10 min. Diagnostic mutant and wild-type amplified bands weredetected on a 2.0% agarose gel post visualization with ethidium bromide.Semi-quantitative PCR was performed using 1 μg of genomic DNA. The sameprimers and thermocycling parameters were applied as above except 28cycles were performed for Step 2. The ratios of these PCR products werecompared against a proportional titration curve of mutant and wild-typeamplified bands (FIG. 10a).

[0107] Bone marrow sampling. Mouse bone marrow was obtained from8-12-week-old animals from each group (−/−, +/+) sacrificed with CO₂.The marrow cell suspensions were flushed from femurs and tibias,filtered with 100-mesh nylon cloth (Sefar America, Inc., Kansas City,Mo.), and stored on ice until use.

[0108] Flow cytometric analysis. Flow cytometry was used to quantify thecell cycle status in the stem cell compartment. Bone marrow nucleatedcells were labeled with anti-lineage antibodies [CD3, CD4, CD8, B220,Gr-1, Mac-1 (Caltac, Burlingame, Calif.), TER-119 (Pharmingen, SanDiego, Calif)] and stem cell marker (Sca-1) (Pharmingen). An enrichedstem cell phenotype (Sca-1⁺Lin⁺) and a progenitor phenotype (Sca-1⁺Lin⁺)were gated, and a DNA dye, To-pro-3, was used to stain theantibody-bound cells simultaneously to measure the cycling cellpercentage in the populations. To measure stem cell quiescence, cellswere stained with lineage antibodies, incubated with 1.67 μmol/L DNAdye, Hoechst 33342 (Hst), and 1 μg/mL RNA dye, Pyronin Y (PY), and theratio of G0 vs. G1 was then measured in the Lin⁻ population (Gothot etal. “Functional heterogeneity of human CD34(+) cells isolated insubcompartments of the G0/G1 phase of the cell cycle” Blood90:4384-4393, 1997; incorporated herein by reference). To detectapoptotic cells, Annexin-V in conjunction with the DNA dye, 7-AAD (Fadoket al. “Exposure of phosphatidylserine on the surface of apoptoticlymphocytes triggers specific recognition and removal by macrophages” J.Immunol. 148:2207-2216, 1992; Shen et al. “Intrinsic humanimmunodeficiency virus type 1 resistance of hematopoietic stem cellsdespite coreceptor expression” J. Virol. 73:728-737, 1999; each of whichis incorporated herein by reference), was used to stain Sca-1⁺, Lin⁻, orLin⁺ cells, which were then analyzed by flow cytometry. Cells excluding7-AAD and binding Annexin-V were considered apoptotic.

[0109] Colony forming assay. Bone marrow nuclear cells were cultured in0.8% methylcellulose, 30% fetal bovine, 1% bovine serum albumin, 0.1 mM2-mercaptoethanol, 2.0 mM L-glutamine of α-MEM semi-solid matrix culturemedium (StemCell Technologies, Inc., Vancouver, Canada) with cytokinecombinations of 50 ng/ml mu-SCF (R & D System Inc., Minneapolis, Minn.),10 ng/ml mu-IL-3 (Genzyme, Cambridge, MA), 10 ng/ml hu-IL-6 (Genzyme),and 3 U/ml hu-Epo (Amgen). Cells were placed at 10,000 to 20,000/200ul/well into 48-well plates and placed at 37° C., 5% CO₂. After 10 days,myeloid and erythroid colonies were scored, totaled and reported asCFCs.

[0110] Long-term culture with limiting dilution. To quantify the stemcells, we adapted the cobblestone area forming cell (CAFC) assay(Ploemacher et al. “An in vitro limiting-dilution assay of long-termrepopulating hematopoietic stem cells in the mouse” Blood 74:2755-2763,1989; incorporated herein by reference) with minor modifications asfollows. To prepare stromal layers, murine bone marrow nucleated cellswere cultured at 33° C. in long term culture (LTC) medium [α-MEM with12.5% house serum, 12.5% fetal bovine serum, 0.2 mM 1-inositol, 20 mMfolic acid, 104 M 2-mercaptoethanol, 2 mM L-glutamine, and 10⁻⁶ Mhydrocortisone]. After 2 weeks, confluent stromal layers weretrypsinized, irradiated (15 Gy), and subcultured in 96-wellflat-bottomed plates at a density of 2.5×10⁴/well. Cultures were thenseeded with serially diluted single-cell suspensions of femoral marrowin the same medium. Marrow pooled from two to ten animals of each typewas seeded at 2-fold dilutions (105 - 1562 cells/well) for nucleatedbone marrow cells. Cultures were very gently re-fed with 50:1 mediumafter semidepletion weekly and the CAFCs and/or blast colonies(Ploemacher et al. “Use of limiting-dilution type long-term marrowcultures in frequency analysis of marrow-repopulating and spleencolony-forming hematopoietic stem cells in the mouse” Blood78:2527-2533, 1991; Muller-Sieburg et al. “Genetic control of thefrequency of hematopoietic stem cells in mice: mapping of a candidatelocus to chromosome 1” J. Exp. Med. 183:1141-1150, 1996; each of whichis incorporated herein by reference) were scored until the 6^(th) week.To measure LIC-IC, methylcellulose medium for CFC (see above) wasoverlaid into the wells at week 5 and the colonies were counted at day10. A limiting dilution software (“Maxrob”, provided by Julian Down,BioTranspant Inc.) was used to calculate the frequency of CAFC orLTC-IC.

[0111] 5-FU exposure in vivo. The anti-metabolite, 5-FU, was used tofunctionally test the cycling status of primitive hematopoietic cells invivo. A single injection of 5-FU i.v. at a dose of 200 mg/kg wasadministered and cells for long-term culture with limiting dilution andcolony forming assay were obtained one day after the injection.

[0112] Serial bone marrow transplantation. Serial bone marrowtransplantation (Harrison “Competitive repopulation: a new assay forlong-term stem cell functional capacity” Blood 55:77-81, 1980; Harrisonet al. “Loss of proliferative capacity in immunohemopoietic stem cellscaused by serial transplantation rather than aging” J Exp. Med147:1526-1531, 1978; Harrison et al. “Effects of transplantation on theprimitive immunohematopoietic stem cell” J Exp. Med. 172:431-437, 1990;each of which is incorporated herein by reference) was used to evaluatethe ability of stem cells to self-renew. Male mice (8-12 weeks old) wereused as marrow donors and the marrow cells prepared as above. Femalerecipient mice (8-10 weeks old, 129/SV, Jackson laboratory) werelethally irradiated using a Mark 1-Model 25 ¹³⁷Cesium Irradiator (JLShepherd and Associates, San Fernando, Calif.) with 10 Gy whole bodyirradiation (WBI) at 5.96 Gy/min. One to two million nucleated cells in1 ml 199 Medium were injected intravenously through 27 gauge needlesinto the lateral tail veins of warmed recipients. Recipient mice weremonitored daily for survival until next transplant. The mice weresacrificed at 1-4 months and bone marrow cells were prepared from thosesacrificed mice and injected into new female recipients. This processwas repeated for 4 sequential transplants with survival frequencyplotted for each group. Long-term culture with limiting dilution andcolony forming assays described above were performed on the donor cellsof each transplant to quantify the frequencies of stem and progenitorcells.

[0113] Short-term radiation-protection assay. 10⁵ marrow nucleated cellsfrom the fourth transplant were transplanted into lethally irradiatedfemale mice as described above, and animal survival frequency wasplotted for each group after 30 days. Results were analyzed using alog-rank nonparametric test and expressed as Kaplan-Meier survivalcurves.

[0114] Competitive long-term repopulation. Equal numbers of bone marrownucleated cells from p27+/+ and p27−/− mice were mixed and transplantedinto the lethally irradiated recipients as described in the serialtransplantation section. Blood was collected at 6 and 9 months forsemi-quantitative p27 PCR analysis. After 12 months, mice weresacrificed and bone marrow nucleated cells were prepared for PCRanalysis and hematopoietic cell culture (CFC, CAFC and LTC-IC; see theCFC and long-term culture sections). Individual colonies from the CFCculture or individual CAFC/LTC-ICs from different wells were isolated bymicropipette and analyzed by PCR for p27.

[0115] Results

[0116] Mice engineered to be p27−/− have a normal stem cell pool, but anenlarged progenitor cell pool

[0117] We first assessed the impact of p27 deletion on differenthematopoietic cell compartments by quantifying the functionalpopulations of progenitor cells (using methylcellulose colony-formingcell (CFC) assays and of more primitive cells (using long-termcobblestone area-forming cell (CAFC) assay) (Ploemacher et al. “An invitro limiting-dilution assay of long-term repopulating hematopoieticstem cells in the mouse” Blood 74:2755-2763, 1989; Ploemacher et al.“Use of limiting-dilution type long-term marrow cultures in frequency Eanalysis of marrow-repopulating and spleen colony-forming hematopoieticstem cells n the r'mouse” Blood 78:2527-2533, 1991; each of which isincorporated herein by reference). The latter assays linearly correlatewith in vivo repopulating potential and were used here as a functionalstem cell assay.

[0118] We observed a marked contrast between p27 −/− mice and p21 −/−mice. In the p21 −/− animals, stem cell populations were doubled andprogenitor populations unchanged in previous studies (Cheng et al.“Hematopoietic stem cell quiescence maintained by p21(cip1/waf1)”Science 287:1804-1808, 2000; Mantel et al. “Involvement ofp21cip-1 and p27kip-1 in the molecular mechanisms of steelfactor-induced proliferative synergy in vitro and of p21cip-1 in themaintenance of stem/progenitor cells in vivo” Blood 88:3710-3719, 1996;each of which is incorporated herein by reference), whereas p27 −/−animals had an increase in progenitors but no change in stem cellnumbers. Decreased numbers of CAFC per nucleated cell in the p27−/−animals were noted compared to +/+ animals (33% reduction of CAFCfrequency in −/− animals, n=7, p=0.0391). However, normalization of thevalues for the overall increase in marrow cellularity (Table 5) in thep27−/− mouse (4.40 vs, 2.83×10⁷/femur pair; p=0.0072; n=6) indicatedthat the number of stem cells per hematopoietic organ (2 femurs/harvest)was not significantly different from the +/+ control (p=0.3861, n=7)(FIG. 6a). However, the progenitor population was significantlydifferent, with an increased CFC population in p27−/− versus +/+ animals(p=0.0006, n=5) (FIG. 6b). Therefore, a disproportionate increase inprogenitor populations and overall cellularity diluted the stem cellfraction, but the absolute number of stem cells was unchanged fromcontrol. TABLE 5 Comparison of total cell number per bone marrow harvestindicates a higher marrow cellularity in the p27 −/− mice Experiment #p27 +/+ p27 −/− 1 3.86 4.26 2 1.63 3.03 3 2.2 3.2 4 3.89 6.9 5 2 2.91 63.4 6.07 Mean 2.83 4.40 p value 0.0072

[0119] An altered cell cycle profile of progenitor cells, but not stemcells in the absence of p27

[0120] To directly measure cell cycle parameters of primitive cellpopulations in the p27+/+ and −/− animals, flow cytometric analysis wasperformed. Because hematopoietic stem cells have been shown to bepositive for the stem cell marker Sca- 1 and negative for lineagemarkers (Spangrude et al. “Purification and characterization of mousehematopoietic stems cells [published erratum appears in Science Jun. 2,1989; 244(4908):1030]” Science 241:58-62, 1988; incorporated herein byreference), we reasoned that lineage marker-expressing cells in theSca-1+ population reflected a population of maturing lineage-committedprogenitors. We confirmed this by testing for CAFC and detected adecrease of 9-20 times in Lin ⁺Sca-1⁺ cells compared to Lin-Sca-1⁺cells. Therefore, flow cytometry was used to separate the enriched stemcell (Sca-1⁺Lin⁻) and progenitor cell (Sca-1⁺Lin⁻) pools from marrownucleated cells and the cell cycle status (S+G2/M percentage) wasanalyzed by simultaneously staining with the DNA dye, To-pro-3. Weobserved a similar S+G2/M percentage of Sca-1⁺Lin⁻ in the p27+/+ and −/−animals (p=0.3591, n=6), but a higher S+G2/M percentage of Sca-1⁺Lin⁺ inthe p27−/− animals (FIG. 7a and 7 b, p=0.0215, n=7). To furtherdistinguish a quiescent fraction (G0) versus G1 in the stem cell pool,an RNA dye, Pyronin Y, was used to stain the marrow nucleated cellswithin a stringent gate of Lin⁻ cells in conjunction with a DNA dye,Hoechst 33342 (Cheng et al. “Hematopoietic stem cell quiescencemaintained by p21 (cip1/waf1) ” Science 287:1804-1808, 2000; Gothot etal. “Functional heterogeneity of human CD34(+) cells isolated insubcompartments of the G0/G1 phase of the cell cycle” Blood90:4384-4393, 1997; each of which is incorporated herein by reference).No difference was observed between p27−/− and +/+ cells (FIG. 7c) unlikethe p21−/− stem cells in which a significantly lower fraction ofquiescent (G0) cells had been previously found (Cheng et al.“Hematopoietic stem cell quiescence maintained by p21 (cip1/waf1)”Science 287:1804-1808, 2000; incorporated herein by reference). Thesedata indicate an unperturbed cell cycle status of stem cells, but anincreased fraction of progenitor cells in active cycle in the absence ofp27.

[0121] Functional evaluation of cell cycle status was performed byexposing animals to the cell cycle-dependent anti-metabolite5-fluorouracil (5-FU), which selectively kills cycling cells (Lemer etal. “5-Fluorouracil spares hematopoietic stem cells responsible forlong-term repopulation” Exp. Hematol. 18:114-118, 1990; Berardi et al.“Functional isolation and characterization of human hematopoietic stemcells” Science 267:104-108, 1995; each of which is incorporated hereinby reference). Littermate −/− or +/+ mice were injected with 200 mg/kgof 5-FU or phosphate-buffered saline alone; marrow was harvested one daylater, and long-term co-culture (CAFC) and colony forming cell (CFC)assays performed (FIG. 8). No difference in the yield of CAFC was notedbetween +/+ or −/− animals suggesting similar proliferative kinetics inthe primitive or stem cell compartment (p=0.2852, n=6). However, asignificant reduction of CFC was observed in the p27−/− group comparedto the +/+ group controls (82.7% versus 50.6%, p=0.0044, n=5) (FIG. 8).The proliferative fraction of cells in the progenitor pool was thereforelarger in those animals lacking p27, providing a basis for the expandedsize of the progenitor cell population.

[0122] An unchanged apoptotic fraction of hematopoietic cells in theabsence of p27

[0123] Under homeostatic conditions, an enlarged cell population in vivomay be caused by increased cell proliferation, decreased cell death, orboth. To assess whether or not altered apoptosis contributed to theexpanded progenitor compartment, we evaluated cells by Annexin-Vstaining (Fadok et al. “Exposure of phosphatidylserine on the surface ofapoptotic lymphocytes triggers specific recognition and removal bymacrophages” J. Immunol. 148:2207-2216, 1992; incorporated herein byreference) and could detect no difference in apoptosis in either theSca-1+Lin⁻stem cell pool or Sca-1^(+Lin) ⁺ progenitor cells betweenp27−/− and +/+ littermate control mice (mean 2.5±0.8 vs. 2.5±0.8 and7.3±2.5 vs. 7.2±2.2, respectively; n=4) (Table 6) TABLE 6 The expansionof progenitor cells in the p27 −/− mice is not due to altered apoptoticeffect Sca-1⁺Lin⁻ Sca-1⁺Lin⁺ Experiment # p27 +/+ p27 −/− p27 +/+ p27−/− 1 2.32 3.27 4.27 6.77 2 3.47 1.38 6.27 4.81 3 2.76 2.87 9.66 10.1 41.62 2.28 9 6.94 Mean 2.5425 2.45 7.3 7.155 SD 0.776332 0.8210972.496758 2.18834

[0124] Reasoning that steady state of the stem cell compartment may beunperturbed yet other physiologic functions affected under stress,sequential bone marrow transplant was performed. Bone marrow from p27+/+or −/− male animals in each genotype was transplanted into 10 lethallyirradiated female mice. One to four months after engraftment, 1-2×10⁶bone marrow cells from the transplanted recipients were used as donorcells for a lethally irradiated host and the same procedure was repeatedsequentially. Chimerism was determined as ˜100% donor derived after eachtransplant by semi-quantitative Y chromosome-specific (Sry) PCR (Mulleret al. “ES cells have only a limited lymphopoietic potential afteradoptive transfer into mouse recipients”Development 118:1343-1351, 1993;incorporated herein by reference) and p27 genotyping PCR (data notshown). There was no difference in bone marrow homing among p27−/− stemcells compared with +/+ controls as assessed by carboxyfluoresceindiacetate succinimidyl diester (CFSE) (Weston et al. “New fluorescentdyes for lymphocyte migration studies. Analysis by flow cytometry andfluorescence microscopy” J. Immunol. Methods 133:87-97, 1990;Grzegorzewski et al. “Recombinant transforming growth factor beta 1 andbeta 2 protect mice form acutely lethal doses of 5-fluorouracil anddoxorubicin” J. Exp. Med. 180:1047-1057, 1994; incorporated herein byreference) staining of Sca-1^(+Lin) ⁻ cells (data not shown). Stem cellquantitation was performed by CAFC analysis following eachtransplantation. A comparable decay rate in CAFC was noted in eachgroup, indicating that stem cell renewal was equivalent in the p27−/−and +/+ animals (FIG. 9a).

[0125] Interestingly, however, the progenitor cell pool from the p27−/−animals was capable of expansion and relative regeneration after serialtransplantation when wild-type progenitors were markedly depleted (FIG.9b). Furthermore, the functional capacity of these cells was evident inimproved animal survival in a short-term radiation-protection assay(FIG. 9c), even after the fourth serial transplant when stem cells wereno longer detectable. These data demonstrate markedly altered cellkinetics among progenitors, but not stem cells, in the absence of p27.This contrasts dramatically with the increased stem cell pool andunaffected progenitor population in the p21−/− setting (Cheng et al.“Hematopoietic stem cell quiescence maintained by p21 (cip1/waf1)”Science 287:1804-1808, 2000; each of which is incorporated herein byreference).

[0126] Preferential outgrowth of p27−/− stem cell descendent cellsfollowing long-term engraftment

[0127] We next tested the role of p27 and thereby the role of progenitorcell cycle inhibition in the context of long-term engraftment. Weperformed a competitive transplantation in which −/− and +/+ bone marrownucleated cells were admixed 1:1 and transplanted into an irradiatedwild-type recipient. It should again be noted that the representation ofstem cells in the −/− nucleated cell preparations is proportionatelylower than in +/+ controls. The admixture of the stem cell population istherefore uneven, with 40% derived from −/− marrow. Aftertransplantation, semiquantitative PCR of p27 was used to monitor eachgenotype in populations of bone marrow and blood cells over a one-yearinterval. It has been shown in other settings that the proportion ofstem cells from a normal host is reflected in a similar proportion oftotal bone marrow cells and blood cells (Harrison “Competitiverepopulation: a new assay for long-term stem cell functional capacity”Blood 55:77-81, 1980; Szilvassy et al. “Quantitative assay fortotipotent reconstituting hematopoietic stem cells by a competitiverepopulation strategy” Proc. Natl. Acad. Sci. USA 87:8736-8740, 1990;each of which is incorporated herein by reference). This is the basisfor the competitive repopulation experiments performed in congenic miceas a tool for measuring stem cell populations. However, in the contextof altered cell cycle regulation by p27 deficiency, proportionaterepresentation in various cellular compartments was strikingly altered.

[0128] Even though the fraction of −/− stem cells transplanted wasapproximately 40%, after six months the fraction of p27−/− cells in theblood reached levels of ˜80% and was sustained at elevated levels (FIG.10a). In addition, the fraction of −/− marrow nucleated cells(predominately a progenitor cell population and its descendents)was >80% at the time of euthanasia at 11 or 12 months (FIG. 10b). CAFC,LTC-IC, and CFC analyses were performed on the marrow specimens.Individual CAFC, LTC-IC, or CFC were then isolated by micropipette forPCR analysis of the p27 genotype. The data confirmed that the proportionof genotypically −/− cells in the CAFC, LTC-IC, or stem cell populationwas comparable to or less than what was transplanted. In contrast, theCFC or progenitor cell population demonstrated a relativeoverrepresentation of the −/− genotype (FIG. 10c). The fraction of stemcells transplanted thereby disproportionately contributed to progenitorcell population, which in turn disproportionately contributed to theblood cell population in the absence of p27. Feedback governing cellpool size therefore is skewed in the absence of p27, permittingovergrowth of progenitors and their descendents in a competitivesituation (FIG. 10d).

[0129] Discussion

[0130] These data support highly differentiation stage-specificregulatory roles for distinct members of the CDKI Cip/Kip family (Table7). TABLE 7 Distinct impact of p21 and p27 on hematopoiesis p21 −/− p27−/− Stem cells Increased number Normal number Increased cycling Normalcycling Accelerated exhaustion Normal exhaustion under under stressstress Progenitor cells Normal number Increased number Normal todecreased Increased cycling cycling Not able to compensate Outcompeted+/+ for stem cell exhaustion progenitors in hemeostasis post transplantand enhanced protection post transplant

[0131] The clearly delineated and apparently exclusive dominance of p27in progenitor cells and p21 in stem cells demarcates a molecularboundary that is unique as far as we know. Cytokine receptors (Berardiet al. “Functional isolation and characterization of human hematopoieticstem cells” Science 267:104-108, 1995; incorporated herein byreference), chemokine receptors (Shen et al. “Intrinsic humanimmunodeficiency virus type 1 resistance of hematopoietic stem cellsdespite coreceptor expression” J. Virol. 73:728-737, 1999; incorporatedherein by reference), adhesion molecules (Becker et al. “Adhesionreceptor expression by hematopoietic cell lines and murine progenitors:modulation by cytokines and cell cycle status” Exp. Hematol. 27:533-541,1999; Roy et al. “Expression and function of cell adhesion molecules onfetal liver, cord blood and bone marrow hematopoietic progenitors:implication of anatomical localization and developmental stage specificregulation of hematopoiesis” Exp. Hematol. 27:302-312, 1999;incorporated herein by reference), and transcription factors (Cheng etal. “Temporal mapping of gene expression levels during thedifferentiation of individual primary hematopoietic cells” Proc. Natl.Acad. Sci. USA 93:13158-13163, 1996; Shivdasani et al. “Thetranscriptional control of hematopoiesis [see comments]” Blood87:4025-4039, 1996; Tenen et al. “Transcription factors, normal myeloiddevelopment, and leukemia” Blood 90:489-519, 1997; Test et al.“Expression of growth factor receptors in unilineage differentiationculture of purified hematopoietic progenitors” Blood 88:3391-3406, 1996;each of which is incorporated herein by reference) have been shown to beexpressed in overlapping populations of precursor populations. Whereasother regulatory molecules may contribute to the differentialsensitivity of stem cells and progenitors to proliferative signals, cellcycle control is highly divergent at the level of the G1-S checkpoint.The distinction between the participating CDKIs may explain in part thehighly dichotomous proliferative capability of stem cells as compared tothe progenitor cells that characterize the hematopoietic and otherdifferentiation systems. Additionally, it provides specific targets forselective manipulation of stem cell versus progenitor cell compartments.To the extent that hematopoiesis mimics other stem and progenitorpopulations in tissue development, this distinction may point to usefulstrategies for altering specific precursor pools in size and activity.

[0132] The observation that competition between p27−/− and +/+ cellsresults in overrepresentation of the −/− progenitor and blood cellsindicates the critical function of inhibition in dictating homeostasisin the later phases of hematopoiesis. The importance ofpro-proliferative cues for hematopoiesis has been demonstrated(Carver-Moore et al. “Low levels of erythroid and myeloid progenitors inthrombopoietin-and-c- mpl-deficient mice” Blood 88:803-808, 1996;incorporated herein by reference), but the crucial role of inhibitors ofproliferation is demonstrated in the p27−/− mice in this study forprogenitors and in p21−/− for stem cells elsewhere. Where there is aninability to exert the cell cycle inhibition mediated by thesemolecules, disruption of normal population kinetics occurs. However, theproliferation that does occur in the p21−/− and p27−/− mice is not asoverwhelming as has been observed, for example, with disruption by aninhibitory cytokine such as TGF-β (Shull et aL “Targeted disruption ofthe mouse transforming growth factor-beta 1 gene results in multifocalinflammatory disease” Nature 359:693-699, 1992; incorporated herein byreference). Although the p27−/− animals have slightly higher bloodcounts than +/+ controls (Table 8), neither the p27- nor p21-deficientanimals develop leukemia or gross polycythemia as indicated by cellcounts, morphology, and phenotypic analysis by flow cytometry (data notshown). Therefore, other negative regulators must be active beyond acertain threshold of cell expansion. It is within physiologic ranges ofthe hematopoietic compartment size that p27 and p21 appear to exertdominant roles in modulating cell dynamics. TABLE 8 Comparison of bloodcell counts indicates slightly higher leukocyte counts in p27 −/− mice(n = 10, Mean ± SD) without significant differences in other mature cellpopulations. WBC (× 10³/ul) RBC (× 10⁶/ul) PLT (× 10⁶/ul) p27 +/+ 6.89 ±2.11 8.26 ± 1.41 635.40 ± 105.64 p27 −/− 9.08 ± 3.12 8.37 ± 1.09 723.10± 172.74 p value 0.0293 0.8193 0.0563

[0133] The ability of a minority population of p27−/− stem cells topredominate in the progenitor and mature blood cell compartmentsindicates the potential efficacy of using p27 to enhance the efficiencyof small numbers of stem cells. A controlled reduction in p27 might makeit possible to effect a marked alteration in a substantially largerfraction of blood cells, particularly in the settings where smallnumbers of stem cells may be transduced with a therapeutic gene. Theabsence of untoward effect in vivo demonstrated in the p27−/− mouseprovides conceptual support. The ability of this genetic alteration toincrease the size of other, non-hematopoietic tissues in vivo (Kiyokawaet al. “Enhanced growth of mice lacking the cyclin-dependent kinaseinhibitor function of p27(Kip1)” Cell 85:721-732, 1996; Fero et al. “Asyndrome of multiorgan hyperplasia with features of gigantism,tumorigenesis, and female sterility in p27 (Kip1)-deficient mice” Cell85:733-744, 1996; Nakayama et al. “Mice lacking p27(Kip1) displayincreased body size, multiple organ hyperplasia, retinal dysplasia, andpituitary tumors” Cell 85:707-720, 1996; each of which is incorporatedherein by reference) indicates that controlled manipulation of p27 mayalso be relevant for the expansion or possible regeneration of othertissue types.

Example 3 Increased numbers of stem cells following ex vivo treatmentwith p21 anti-sense

[0134] Materials and Methods

[0135] Cells and cell culture. Cells were obtained from umbilical cordblood after normal full-term deliveries, from bone marrow harvests ofhealthy adult volunteers and from mobilized peripheral blood of normaldonors in accordance to procedures approved by the Institutional ReviewBoard of the Massachusetts General Hospital. Samples were diluted in PBSand enriched for mononuclear cells by centrifugation on Ficoll/Paque.CD34⁺ were enriched by immunomagnetic selection in according to themanufacturer's instructions (Miltenyi Biotec, Bergisch-Gladbach,Germany) with a purity in the selected product always of 95%. CD34⁺38⁻cells were further enriched after staining with CD34-fluoresceinisothiocyanate (FITC) and CD38-phycoerythrin (PE) (Becton Dickinson, SanJose, Calif.) by fluorescence-activated sorting (FACS Vantage, BectonDickinson).

[0136] Human embryonic kidney derived 293T cells were grown inDulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS, 100U/ml penicillin, 100 U/ml sreptomycin, and 2 mM L-glutamine (GIBCO,BRL). CMK cell line was grown in RPMI supplemented with 10% FCS, 100U/ml penicillin, 100 U/ml sreptomycin, and 2 mM L-glutamine (GIBCO,BRL).

[0137] Lentviral vectors and constructs. cDNA encoding full lengthp21^(cip1) was subcloned as antisense into the BamHI cloning site of thelentiviral vector pHR-CMV-GFP (Miyoshi et al. Proc. Natl. Acad. Sci. USA94(19):10319-10323, 1997; incorporated herein by reference). The controlvector contains the cDNA encoding the green flourescent protein.

[0138] Lentviral production and transduction. The lentiviral vectorcontaining p21cip1antisense (p21-AS-V) and the control vector (GFP-V)were cotransfected into 293T cells with pCMV encoding the gag and polproteins, and pCMV-VSV-G, a plasmid encoding the vesicular stomatitisvirus G-glycoprotein (VSV-G), using the Geneporter lipofection method inaccording to the manufacturer's instructions (Gene Therapy Systems, SanDiego, Calif.). Supernatants containing pseudotyped lentiviruses werecollected at 72 hrs after the beginning of transfection and were usedfor the transduction of human CD34⁺ and CD34⁺38⁻ hematopoietic cells.

[0139] CD34⁺ and CD34⁺38⁻ cells were cultured in Iscove's modifiedDulbecco's medium (IMDM) containing 10% fetal calf serum (FCS; Sigma,St. Louis, Mo.) (IMDM 10), 100 U/ml penicillin, 100 U/ml sreptomycin and2 mM L-glutamin (GIBCO, BRL) supplemented with stem cell factor (SCF [50ng/ml]), Flt-3-ligand (Flt-3-L [50 ng/ml]), Thrombopoietin (TPO [25ng/ml]) and Interleukin-3 (IL-3 [10 ng/ml]) (R&D Systems, Minneapolis,Minn.) for 24 hrs on Retronectin (Takara, Japan) coated wells. Afterthis prestimulation, two third of the culture medium was dicarded andreplaced with the viral containing supernatant plus Polybrene (finalconcentration 4 ,μg/ml, Sigma). The cells with the viral supernatantwere spinoculated at 1700 revolutions per minute for 30 minutes,incubated at 37° C. and 5% C0₂ for an additional 20 hrs, than washed andplated on fresh Retronectin coated wells in IMDM 10 plus cytokinesovernight. A second transduction was performed on the following dayusing the same procedure. Four days after beginning the transduction thetransduction efficiency was measured by flow cytometric analysis (FACSCalibur, Becton Dickinson) for GFP⁺ cells.

[0140] Colony forming assay. Transduced CD34⁺ and CD34⁺38⁻ cells werecultured in 0.8% methylcellulose, 30% fetal bovine serum, 1% bovineserum albumin, 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine of α-MEMsemi-solid matrix culture medium supplemented with cytokines (SCF [50ng/ml], IL-3 [10 ng/ml], IL-6 [10 ng/ml] and Erythropoietin (EPO) [4U/ml]) (StemCell Technologies Inc., Vancouver, Canada). Cells wereplated at 500 cells/ml into 24-well plates and placed at 37° C. and 5%CO₂. At day 10, colonies were scored, totaled and reported as CFCs.

[0141] Long-term culture with limiting dilutions. To quantify the stemcells in the transduced CD34⁺ and CD34⁺38⁻ cell population, we adaptedthe cobblestone area forming cell (CAFC) assay (Ploemacher et al. “An invitro limiting-dilution assay of long-term repopulating hematopoieticstem cells in the mouse” Blood 74:2755-2763, 1989; Ploemacher et al.“Use of limiting-dilution type long-term marrow cultures in frequencyanalysis of marrow-repopulating and spleen colony-forming hematopoieticstem cells in the mouse” Blood 78:2527-2533, 1991; each of which isincorporated herein by reference) with minor modifications as follows.To prepare stromal layers human bone marrow nucleated cells werecultured at 33° C. in long-term culture (LTC) medium (α-MEM with 12.5%horse serum, 12.5% fetal bovine serum, 0.2 mM I-inositol, 20 mM folicacid, 0.1 mM M 2-mercaptoethanol, 2 mM L-glutamine, and 1 μMhydrocortisone; StemCell Technologies). After 4-8 weeks the confluentstromal layers were trypsinized, irradiated (15 Gy), and subcultured in96-well plates at a density of 2.5×10⁴ cells per well. The transducedCD34⁺ and CD34⁺38⁻ cells were then seeded with 2-fold diluted singlecell suspensions in the same LTC-medium. Half of the medium was replacedweekly and the CAFC's were scored until the 6th week (Ploemacher et al.“Use of limiting-dilution type long-term marrow cultures in frequencyanalysis of marrow-repopulating and spleen colony-forming hematopoieticstem cells in the mouse” Blood 78:2527-2533, 1991; incorporated hereinby reference). To measure LTC-initiating cells (LTC-IC) the semisolid,cytokine containing methylcellulose medium for CFC (s.a.) was overlaidinto the wells at week 5 and the colonies were counted at day 10. Alimiting dilution analysis software program (Maxrob, kindly provided byJulian Down, BioTransplant Inc.) was used to calculate the frequency ofLTC-ICs in the cell population.

[0142] Liquid culture. To examine the affect of p21 -antisense on thedifferentiation and expansion of hematopoietic cells transduced CD34⁺and CD34⁺38⁻ cord blood cells were cultured in IMDM 10 supplemented withSCF [50 ng/ml], Flt-3 [50 ng/ml] and TPO [10 ng/ml]. Weekly the mediumwas replaced and half of the cells were taken for further analysis. Tomeasure the proportion of primitive cells in the liquid culture, cellswere stained with CD34-PerCP and CD38-APC (Becton Dickinson), incubatedwith propidium iodid to distinguish between viable and dead cells, andanalyzed by flow cytometry.

[0143] NOD/SCID repopulation assay. To evaluate the repopulation abilityof the transduced human CD34⁺ cells we used a NOD/SCID repopulationassay. NOD/SCID mice (Jackson Laboratories, Bar Harbor, Me.) werehandled under sterile conditions and maintained under mircoisolaters.Transduced CD34⁺ umbilical cord blood cells were transplanted by tailvein injection into sublethal irradiated (3.5 Gy) 8-week-old mice alongwith 1.5×10⁶ irradiated (20 Gy) nonrepopulating human bone marrowmononuclear cells. Every two weeks after the first month ca. 200 μl ofperipheral blood was obtained from each recipient mice by tail bleeds.The blood was stained with CD45-PerCP, CD38-APC (Becton Dickinson)antibodies, treated with a lysis buffer (ACK Lysis buffer), incubatedwith propodium iodid to distinguish between viable and dead cells, andanalyzed by flow cytometry to detect human derived hematopoieticprogenitors (FACS-Calibur, Becton Dickinson). Mice were sacrificed 6 to12 weeks after transplantation. Bone marrow from femurs and tibiae ofeach mouse were flushed into IMDM containing 10% FCS and analyzed byflow cytometry (FACS-Calibur, Becton Dickinson).

[0144] Flow cytometric analysis. Flow cytometry was used to estimate thetransduction efficiency and content of stem cells in the transduced cellpopulation 4 days after the beginning of the transduction. Cells werestained with CD34-PerCP and CD38-APC (Becton Dickinson) and incubatedwith propidium iodid shortly prior to the flow cytometric analyis, todistinguish between viable and dead cells.

[0145] To quantify the repopulation ability of the transplantedtransduced CD34⁺ cord blood cells in the peripheral blood and the bonemarrow of the tranplanted animals (NOD/SCID repopulation assay s.a.)bone marrow nucleated cells were labeled with the human leukocyteantibody CD45-PerCP, stem cell markers (CD34-PE and CD38 APC), andlineage antibodies (CD3-APC, CD11-APC, CD14-APC, CD19-APC (BectonDickinson), CD41 PE and Glycophorin A. The viability of the stainedcells were measured by staining with propidium iodid (PI) or 7-AAD 15min prio to the analysis and gating on PI or 7-AAD negative cells. Thestained cell samples were analyzed on a FACScalibur cytometer (BectonDickinson).

[0146] Flow cytometric analysis of the cell cycle status. TransducedCD34⁺ cord blood cells were stained with CD34-PE and CD38-APC (BectonDickinson) followed by an incubation with a DNA-dye Hoechst33342 (Hst,1.67 μmol/l) (Hoechst) and RNA-dye, PyroninY (PY, 1 ,μg/ml) (Gothot etal. “Functional heterogeneity of human CD34(+) cells isolated insubcompartments of the G0/G1 phase of the cell cycle” Blood90:4384-4393, 1997; incorporated herein by reference). The proportion ofcells in G0 as PY^(low) Hoechst^(low) cells was measured in the CD34⁺38⁻cell subpopulation, representing quiescent primitive hematopoieticcells.

[0147] Western blot analysis. To confirm that transduction of thep21-AS-V leads to an decreased expression of p21, CMK cells (2×10⁶) weretransduced with p21-AS-V and the control vector followed by astimulation with TPA (100 nM, Sigma) 24 hrs after the beginning oftransduction to induce p21 -expression. After further 24 hours cellswere lysed in an ELB lysing buffer. Total protein was separated in 12.5%denaturating gel, blotted on a membrane and probed with anti-human p21(clone 6B6, Pharmingen, San Diego, Cailf.).

[0148] Statistical analysis. The significance of the difference betweengroups in the in vitro and in vivo experiments were evaluated byanalysis of variance followed by a two-tailed Student's t-test.

[0149] Results

[0150] Lentiviral expression of p21-antisense in human CD34⁺ cord bloodcells. cDNA encoding full length p21^(cip1) was subcloned intopHR′-CMV-GFP (GFP-V), a lentiviral vector that allows coexpression ofsubdloned cDNAs and green fluorescent protein (GFP) from a single MRNAtranscript (Miyoshi et al. Proc. Natl. Acad. Sci. USA94(19):10319-10323, 1997; incorporated herein by reference), asantisense (p21 -AS-V).

[0151] The transduction efficiency of human CD34⁺ and CD34⁺38- cordblood cells by p21-AS-V was measured by flow cytometric analysis.Independent experiments showed a transduction efficiency of 45-55% forthe control vector (GFP-V) and 25-35% for the p21-AS-V lentiviruses fourdays after the beginning of transduction. At this time point cells wereused for in vitro and in vivo experiments.

[0152] p21-antisense reduces the G₀ fraction of transduced CD34⁺ cordblood cells. To evaluate the ability of p21-antisense to alter cellcycle kinetics we analyzed the cell cycle status of transduced CD34⁺cord blood cells by simultaneously staining with DNA and RNA dyes, whichallows the distinction between cells in G₀ and G₁ (Gothot et al.“Functional heterogeneity of human CD34(+) cells isolated insubcompartments of the G0/G1 phase of the cell cycle” Blood90:4384-4393, 1997; Cheng et al. “Hematopoietic stem cell quiescencemaintained by p21 (cip1/waf1)” Science 287:1804-1808, 2000; each ofwhich is incorporated herein by reference). Cells determined to be inthe G0/G1 phase of the cell cycle based on the Hst fluorescencedistribution can be further fractionated into subcompartments of varyingcellular RNA content by staining with PY. Quiescent cells, in G₀, have alow RNA content. As cells progress through G₁, they accumulate RNA andfinally move to the S/G₂+M phase during which Hst staining increases. In6 independent experiments transduction of p21-antisense decreased theproportion of cells in G₀ in the CD34⁺38⁻ subpopulation of transducedCD34⁺ cordblood cells (7.3% p21-AS-V vs. 16.4% GFP-V; p=0.007),indicating that p21-antisense promotes the entry of quiescent cells intothe cell cycle (FIG. 11). p21-antisense increases primitivehematopoietic cells in transduced CD34⁺ and CD34⁺38⁻ cord blood cells invitro. We next sought to define the impact of p2l -antisense on thedifferentiation status of transduced CD34⁺ and CD34⁺38⁻ cord blood cellsin vitro. Transduced cells were analyzed for their ability to generatecolonies using methylcellulose colony forming (CFC) assays forprogenitor function. Transduced cells were plated four days after thebeginning of transduction in semisolid CFC-medium. Neither CD34⁺(n=4)nor CD34⁺38⁻ cells transduced with p21 -antisense showed a altered totalcolony number compared to cells transduced with the control vector (FIG.12B). Of note, however, the colonies generated by cells expressing p21-antisense showed a higher proportion of colonies with myloid anderythroid cells (CFU-mix) representing more primitive hematopoieticcells than colonies of the control vector transduced cells (CD34⁺: 9.3vs. 2.8 colonies/600 cells, p=0.02; CD34⁺38⁻: 19.2 vs. 7.1 colonies/600cells, p=0.002) (FIG. 12A).

[0153] To quantify the stem cell frequency in the transduced CD34⁺ andCD34⁺38⁻ cell population, we performed long-term cultures with limitingdilutions on primary human bone marrow stroma (LTC-IC-assay). CD34⁺ andCD34⁺38⁻ cells transduced with p21-antisense gave rise to asignificantly higher number of long-term culture initiating cells(LTC-ICs) compared with cells transduced with the control vector,indicating a higher proportion of stem cells in the p21-antisensetransduced cell population (FIG. 13, CD34⁺: 33.5 vs. 19.3 LTC-ICs/100000cells (p=0.04); CD34⁺38⁻: 416 vs. 228 LTC-ICs/100000 cells (p =0.03)).Like in the CFC-assay overexpression of p21 -antisense led to anincrease of primitive hematopoietic cells in comparison to controlvector transduced cells. Thus, p21-antisense expands or preservesprimitive hematopoietic cells measured by functional in vitro assays.

[0154] p21-antisense increases stem cell numbers as measured byrepopulation of NOD/SCID mice. The ability of human cells to engraftmultiply immunodeficient, NOD/SCID, mice has provided an in vivo modelof a stem cell functional phenotype. The abundance of human cells in theblood or bone marrow of engrafted animals correlates with the inputnumber of stem cells. Using this assay, we transplanted human cellstransduced with either control vector or the p21 -antisense vector intoirradiated mice and evaluated human cell engraftment. Cells transducedwith control vector demonstrated minimal engraftment, substantiallydifferent from those transduced with the p21 -antisense vector.Therefore, p21 -antisense enhances the number of stem cells as measuredby this in vivo model of stem cell function. FIG. 14 demonstrates thepercent of human cells detectable in the blood of animals transplantedwith cells exposed to either control (GFP-V) or p21 -anti-sense encoding(p21 -AS-V) vector.

Other Embodiments

[0155] The foregoing has been a description of certain non-limitingpreferred embodiments of the invention. Those of ordinary skill in theart will appreciate that various changes and modifications to thisdescription may be made without departing from the spirit or scope ofthe present invention, as defined in the following claims.

What is claimed is:
 1. A method of expanding a population of cells, themethod comprising steps of: providing at least one cell with less thanwild type p21 activity; and expanding the cell population.
 2. The methodof claim 1, wherein the step of providing comprises: providing a cell;and disrupting p²1 gene.
 3. The method of claim 1, wherein the step ofproviding comprises: providing a cell; and contacting the cell with anagent, wherein the agent inhibits p21 activity.
 4. A method of expandinga population of cells, the method comprising the steps of: providing atleast one cell with less than wild type p27 activity and less than wildtype p21 activity; and expanding the cell population.
 5. The method ofclaim 4, wherein the step of providing comprises: providing a cell; anddisrupting p27 and p21 genes.
 6. The method of claim 4, wherein the stepof providing comprises: providing a cell; and contacting the cell withan agent, wherein the agent inhibits p27 and p21 activity.
 7. The methodof claim 1, wherein the cell is a stem cell.
 8. The method of claim 1,wherein the cell is a hematopoietic stem cell.
 9. The method of claim 1,wherein the cell is a hematopoietic progenitor cell.
 10. The method ofclaim 1, wherein the cell is an erythropoietic cell.
 11. The method ofclaim 1, wherein the cell is a granulopoietic cell.
 12. The method ofclaim 1, wherein the cell is a thrombopoietic cell.
 13. The method ofclaim 1, wherein the cell is a neural cell.
 14. The method of claim 1,wherein the cell is selected from the group consisting of renal cell,gastrointestinal cell, hepatic cell, skin cell, lung cell, muscle cell,and cardiac muscle cell.
 15. The method of claim 1, wherein the cell isan adult-derived stem cell.
 16. The method of claim 1, wherein the cellis an embryonically derived stem cell.
 17. The method of claim 1,wherein the cell is a pluripotent stem cell.
 18. The method of claim 1,wherein the cell is a multi-potential stem cell.
 19. The method of claim1, wherein the cell is a fetal cell.
 20. The method of claim 1, whereinthe cell is an embryonic cell.
 21. The method of claim 1, wherein thecell is a mesenchymal cell.
 22. The method of claim 3, wherein the agentis a protein.
 23. The method of claim 3, wherein the agent is a peptide.24. The method of claim 3, wherein the agent is a polynucleotide. 25.The method of claim 3, wherein the agent is a chemical compound.
 26. Themethod of claim 3, wherein the agent is an antibody or fragment thereof.27. The method of claim 3, wherein the agent is an antisense agent. 28.The method of claim 3, wherein the agent is a triple helix formingagent.
 29. The method of claim 3, wherein the agent is an aptamer.
 30. Acell with less than wild type p21 activity.
 31. A cell with at least onecopy of the p21 gene disrupted.
 32. A cell with both copies of the p21gene disrupted.
 33. A cell with less than wild type p27 activity.
 34. Acell with at least one copy of the p27 gene disrupted.
 35. A cell withat least one copy of the p27 gene and p21 gene disrupted.
 36. The cellof claim 30, wherein the cell is a stem cell.
 37. The cell of claim 30,wherein the cell is a progenitor cell.
 38. A stem cell with increasedcyclin activity.
 39. A progenitor cell with increased cyclin activity.40. A stem cell with increased cyclin-dependent kinase activity.
 41. Aprogenitor cell with increased cyclin-dependent kinase activity.
 42. Apharmaceutical composition comprising a therapeutically effective amountof cells of claim
 30. 43. A pharmaceutical composition comprising atherapeutically effective amount of stem cells of claim
 36. 44. Apharmaceutical composition comprising a therapeutically effective amountof progenitor cells of claim
 37. 45. A pharmaceutical compositioncomprising a therapeutically effective amount of cells of claim 30, anda pharmaceutically acceptable excipient.
 46. A non-human transgenicanimal wherein at least one copy of the p21 gene is altered.
 47. Anon-human transgenic animal wherein both copies of the p21 gene arealtered.
 48. A non-human transgenic animal wherein at least one copy ofthe p27 gene is altered in the hematopoietic cell line.
 49. A non-humantransgenic animal wherein both copies of the p27 gene are altered in thehematopoietic cell line.
 50. A non-human transgenic animal wherein atleast one copy of the p27 gene is altered and at least one copy of p21gene is altered.
 51. A non-human transgenic animal wherein both copiesof p27 gene and both copies of p21 gene are altered.
 52. The non-humantransgenic animal of claim 46 wherein the animal is a mouse.
 53. Thenon-human transgenic animal of claim 46 wherein the animal is a rat. 54.A transgenic plant with at least one copy of the p21 gene altered.
 55. Atransgenic plant with both copies of the p21 gene altered.
 56. A methodof gene therapy, the method comprising steps of: providing at least onecell with less than wild type p21 activity; and altering genome of saidcell.
 57. A method of gene therapy, the method comprising the steps of:providing at least one cell with less than wild type p27 activity; andaltering genome of said cell.
 58. A method of gene therapy, the methodcomprising the steps of: providing at least one cell with less than wildtype p27 activity and p21 activity; altering genome of said cell. 59.The method of claim 56, the method comprising the additional step ofdelivering the cell to an animal.
 60. The method of claim 56, whereinthe cell is a stem cell.
 61. The method of claim 56, wherein the cell isa progenitor cell.
 62. The method of claim 56, wherein the cell is ahematopoietic stem cell.
 63. The method of claim 56, wherein the cell isa hematopoietic progenitor cell.
 64. A method of gene therapy, themethod comprising steps of: providing at least one cell with less thanwild type p27 activity; altering the genome of the cell; andtransplanting into an individual a therapeutically effective amount ofcells wherein the percentage of transplanted altered cells is less than10% of total cells transplanted.
 65. A method of gene therapy, themethod comprising steps of: providing at least one cell with less thanwild type p21 activity; altering the genome of the cell; andtransplanting into an individual a therapeutically effective amount ofcells wherein the percentage of transplanted altered cells is less than10% of total cells transplanted.
 66. A method of gene therapy, themethod comprising steps of: providing at least one cell with less thanwild type p21 and p27 activity; altering the genome of the cell; andtransplanting into an individual a therapeutically effective amount ofcells wherein the percentage of transplanted altered cells is less than10% of total cells transplanted.
 67. The method of claim 64, wherein thepercentage of transplanted altered cells is less than 25% of total cellstransplanted.
 68. The method of claim 64, wherein the percentage oftransplanted altered cells is less than 50% of total cells transplanted.69. The method of claim 64, wherein the percentage of transplantedaltered cells is less than 5% of total cells transplanted.
 70. Themethod of claim 64, wherein number of cells transplanted is sufficientto allow the altered cells to expand in vivo and compete out wild typecells in the individual.
 71. The method of claim 64, wherein number ofcells transplanted is sufficient to allow the altered cells to expand invivo and account for at least 50% of the individual's cells of thetransplanted type.
 72. The method of claim 64, wherein number of cellstransplanted is sufficient to allow altered cells to expand in vivo andaccount for at least 75% of the individual's cells of the transplantedtype.
 73. The method of claim 64, wherein number of cells transplantedis sufficient to allow altered cells to expand in vivo and account forat least 90% of the individual's cells of the transplanted type.
 74. Amethod of tissue regeneration, the method comprising steps of: providingat least one cell with less than wild type p21 activity; andadministering the cell to an individual in need of tissue regeneration.